Sequence, tissue distribution and developmental changes in rat intestinal oligopeptide transporter

Biochi~ic~a et BiophysicaA~ta

ELSEVIER

Biochimica et Biophysica Acta 1305 (1996) 34-38

Short sequence-paper

Sequence, tissue distribution and developmental changes in rat intestinal oligopeptide transporter 1 Ken-ichi Miyamoto a,*, Toshiyuki Shiraga a, Kyoko Morita a, Hironori Yamamoto a, Hiromi Haga a, Yutaka Taketani a, Ikumi Tamai b Yoshimichi Sai b, Akira Tsuji b Eiji Takeda a a Department of Clinical Nutrition, School of Medicine, Tokushima University, Tokushima 770, Japan b Department of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920, Japan

Received 4 July 1995; revised 13 September 1995; accepted 17 October 1995

Abstract

Complementary DNA clones encoding the rat PepT1 small-intestinal oligopeptide transporter were isolated from a jejunal library by cross-hybridization with a rabbit PepT1 cDNA probe. The cDNA sequence indicates that rat PepT1 is composed of 710 amino acids and shows 77% and 83% amino acid sequence identity with rabbit and human PepT1, respectively. Northern blot analysis detected rat PepT1 mRNA in the small intestine and kidney. Intestinal PepT1 mRNA levels were highest in 4-day-old rats, and then decreased reaching the adult level by day 28 after birth. These results indicate that the expressions of PepT1 gene change markedly during development. Keywords: Oligopeptide transporter; Small intestine; (Rat)

Peptide transport in the mammalian small intestine plays a central role in the absorption of dietary proteins and is mediated by a specific transport system localized in the brush border membrane that is distinct from the transport systems described for free amino acids [1]. The peptide transporter accepts di- and tripeptides as substrates and is dependent on the transmembrane H ÷ gradient [2]. The transporter is also important pharmacologically because it mediates the intestinal absorption of orally active fl-lactam antibiotics and other peptide-like drugs [3,4]; indeed, it has been proposed that the transporter has the potential to become an important drug delivery system. Recently, a rabbit cDNA was isolated that encodes a 707-amino acid peptide transporter, termed PepT1 [5]. A human homolog, HPepT1, that shares 81% amino acid sequence identity with rabbit PepT1 was also identified in intestine [6]. The PepTl-mediated transport system accepts small peptides that contain neutral, basic, or acidic amino acids [5]. The

* Corresponding author. Fax: + 81 886 337094. i The nucleotide sequence data from which the amino acid sequence reported in this paper was deduced have been submitted to the DDBJ data base under the accession No. D50664. 0167-4781/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved

SSDI 01 6 7 - 4 7 8 1 ( 9 5 ) 0 0 2 0 8 - 1

structural attributes of the PepT1 substrate binding site that allow such a broad substrate specificity are not known. The PepT1 amino acid sequence shows no significant homology to other known mammalian sequences [5,6]. Comparison with the amino acid sequences of PepT1 from other species, however, may reveal important structural and functional domains. We now describe the molecular cloning, tissue distribution and developmental changes of the rat peptide transporter.

Experimental

animals

and tissue preparations.

Sprague-Dawley rat pups of 2 days old were distributed among mothers to maintain a litter size of 9-10 pups until the time of study. Mothers, weanling and adult rats were maintained on a semisynthetic diet (20% casein) as described previously [8]. Rats were killed with an overdose of ether, the abdomen was opened and the jejunum was removed. The jejunum was opened along its mesenteric side, and mucosal cells were scraped off with a glass slide. Cloning of cDNAs encoding rat PepT1. Rat jejunal polyadenylated RNA was prepared by CsC1 and guanidium isothiocyanate purification followed by oligo(dT)-cellulose affinity chromatography as previously described [7,8]. A cDNA library in vector hgtl0 (4.104 independent recombinants) was constructed from 5 /xg of the polyadenylated

K.-i. Miyamoto et al. / Biochimica et Biophysica Acta 1305 (1996) 34-38

GCG~CTCCTGCT~CCAGTCGCC~TCAGGAGCCTCGGAGCCGCCAC~TGGGGA~

60

M G M TCC~GTCTC~GTT~TT~TACCCA~GAGCATCT~CATCGT~TC~ S K S R G C F G Y P L S I F F I V V

120 N

E

TTCTGTG~GATTCTCCTACTAT~ATGCGAGCTCTCC~TTCTGTACTTCA~C F C E R F S Y Y G M R A L L V L Y F R N

180

TTCCTT~CT~A~ATGACCTCTCCAC~CCA~TACCATACGTT~T~CCCTC~ F L G W D D D L S T A I Y H T F V A L C

240

TACCTGACTCC~TTCTTGGA~TC~ATCGCAGACTCGT~TGGGG~GTTC~GACA Y L T P I L G A L I A D S W L G K F K T

300

AT~TCTCACTATCCATCGTCTACACGATC~ACA~CGTCATCTCAG~A~TC~TT I V S L S I V Y T I G Q A V I S V S S I

360

~TGACCTTACAGACCATGACCACGAC~AGTCCT~C~CCT~C~CACGTAGCA N D L T D H D H D G S P N N L P L H V A

420

C~TCCA~ATC~CCT~CCTGATAGCCC~TACA~A~GCCCTGTG~ L S M I G L A L I A L G T G G I K

480 P

C

V

~TGCATTTGGTGGCGATCAGT~A~AAAAACAGCGAAACC~TTCT~ S A F G G D Q F E E G Q E K Q R N R

F

F

TCCA~T~TATT~CTA~C~A~GCCT~TCTCCACGATCATCACTCCCATA S I F Y L A I N A G S L L S T I I T P

I

CTCAGAGTTCAGCAG~TCCACA~C~C~GCT~TTACCCAC~CCTTT~ L R V Q Q C G I H S Q Q A C Y P L A F

G

GTTCC~CA~TCTCA~GC~T~CCT~TTG~TCCTC~AG~G~TAC720 V P A A L M A V A L I V F V L G S G

540

600

660

M

Y

~G~GTTTCAGCCCCAGGC4~CATCA~GT~C~G~A~C~TTTGCC780 K K F Q P Q G N I M G K V A K C I R F

A

ATCAAAAACA~T~C~CACCG~GT~G~ATT~CC~GA~CAC~GC~AC I K N R F R H R S K A F P K R E H W

L

D

TGGGCT~G~TACGAT~GA~CTCATC~AGA~GA~GTGACG~ W A K E K Y D E R L I S Q I K M

K

V

840

900 V

T

A~T~C~TACATTCCCCTCCCCA~TTGGGCCT~TT~ACCAGCA~TTCCA~ M F L Y I P L P M F W A L F D Q Q G S R

960

~ACAC~C~GC~CGACCA~ACTGGGAAAAT~C~TTGA~TTCA~C~ACI020 W T L Q A T T M T G K I G T I E I Q P D CAGA~CAGACGG~C~CA~TTGAT~TCA~AT~CCCA~GTGGAC~CGTGI080 Q M Q T V N A I L I V I M V P I V D A V G~TATCCGCTCAT~AAAATG~TC~CTTCACCTCCC~G~GA~CCGTT V Y P L I A K C G F N F T S L K K M T

V

~A~TTCC~GCA~CA~CTT~TGGT~C~CA~TCGATI200 G M F L A S M A F V V A A I V Q

D

V

E

I

1140

35

RNA by oligo(dT)-primed cDNA synthesis [7]. Plaques were screened by hybridization under high-stringency conditions with a 32P-labeled rabbit PepT1 polymerase chain reaction product (420 bp) [5]. Five positive clones were isolated, subcloned into the NotI site of pBluescript II SK ÷ (Stratagene), and characterized by restriction mapping with ApaI, PvulI, PstI, EcoRI, or HindlII. Both strands of the cDNA inserts were sequenced by the dideoxy chain termination method with a T7 sequencing kit (Pharmacia). Synthetic oligonucleotides were used as primers to complete the sequence. Sequence reactions and gels were performed at least three times and for both strands of the cDNA. Resolution was improved in some regions by replacing dGTP with deaza dlTP in the nucleotide mix. Northern blot analysis. Total RNA (15 /~g) from various rat tissues was denatured, subjected to electrophoresis on a 1.2% agarose gel containing formaldehyde, transferred to a nylon membrane (Gene Screen, DuPont-New England Nuclear), and hybridized with a 32P-labeled, randomly primed 1.9-kb rat PepT1 cDNA probe [9]. We used a rabbit PepT1 cDNA fragment (nucleotide position + 1 to +420, relative to the transcription start site) to isolate the corresponding full-length rat cDNA [5]. The largest of the five positive rat cDNA clones, clone Pepl 1, contained an insert of 3042 bp, similar to the size of rat PepT1 mRNA as determined by Northern analysis (see below). The nucleotide and deduced amino acid sequences of rat PepT1 are shown in Fig. 1. The first ATG codon lies within a good consensus initiation sequence (a purine(A) at position - 3 and a G at position + 4 ) [10]. The open reading frame continues to the first stop codon (TGA) at base 2182 and encodes a 710-amino acids protein with a calculated molecular mass of 79 052 Da. Hydropathy analysis of the predicted amino acid sequence

A A A A C T C ~ C C A G ~ C C C C A ~ G T T C ~ T C ~ G ~ C A ~ A I 2 6 0 K T L P V F P S G N Q V Q I K V L N I G

W

~C~TGACA~GCCG~TAT~TCCT~ACAGT~CCCAAA~TeAGI320 N N D M A V Y F P G K N V T V A Q M

T T C G A C A A A C A G TGGGC T G A G T A T G T T C TGTTCGC C TC CTTGCTC C T G G T C G T C T G C A T C F D K Q W A E Y V L F A S L L L V V C I

2040

ATA T TTGC C A T T A T G G C C C G A T T C TACAC C T A C A T C A A C C C A G C A G A G A T C G A G G C A C A G I F A I M A R F Y T Y I N P A E I E A Q

2100

TTCGATGAGGATGAGAAGAAAAAGGGCGTAGGGAAGGAAAA CC C G T A T T C C TC G T T G G A A F D E D E K K K G V G K E N P Y S S L E

2160

S

TGGCTTCTAACCGTGGCCATCGGTAATATCATTGTCCTCATTGTGGCTGAGGCAGGCCAC

Q

ACAGACACATTCA~ACT~CGA~TAGACCA~TGAC~GCAT~CG~TTC~CC1380 T D T F M T F D V D Q L T S I N V S S P

~ATCTCCA~GTCACCAC~TA~TCA~AGTT~A~CGGGTCACC~CACACCCTT G

S

P

G

V

T

T

V

A

H

E

F

E

P

G

H

R

H

T

1440

L

CTAGTGTGGGGCCCC~TCTATACCG~T~TAAAAGAC~TCTT~CCAAAA~CAGAG L V W G P N L Y R V V K D G L N Q K P E

1500

~GGGGAG~C~TCAGA~CGTCA~ACCC~C~GA~ATCACCATCAAAATGI560 K G E N G I R F V S T L N E M I T I K M AGT~AAAAGTGTACGAAAATGTCACCAGTCACAGC~CAGC~CTATCAG~CCCT S G K V Y E N V T S H S A S N Y Q F F P

1620

TCTGGCCAAAAAGACTACAC~T~CACCACAGAGAT~CACe~C~CA~TGAT S G Q K D Y T I N T T E I A P N C S S D

1680

~T~TCTTCC~CCTTGACTTC~CAGCGCGTACACCTACGTGATCAG~GTA~CG F K S S N L D F G S A Y T Y V I R S R A

1740

AG~A~CTGCCT~GTG~TTCG~GACATCCCACCC~CAC~CA~IS00 S D G C L E V K E F E D I P P N T V

N

M

GCCC~AGATCCCACAGTACTTCC~C~ACC~C~CGA~T~TC~CTCTGTCACA A L Q I P Q Y F L L T C G E V V F S V T

1860

~ACT~AGTTCTCCTATTCCCAGGCCCCGTCT~CATG~GTCCG~TTCA~CA~A G L E F S Y S Q A P S N M K S V L Q A G

1920

L

L

T

V

A

I

G

N

C C TGTC T C A C A G A C A A A C A T G T G A A G A T P V S Q T N M *

I

I

V

L

I

V

A

E

A

G

CAGAAAGCAAGTGGAGAACATACCAAGTCC

1980 H

AG 2220

C A T T C A C C A T G A C C TCTGCC C A A G G G A C A G G A C CCTC C A C C A C A G A G T C C T T G C C T G G A G A A A G A C TTC A G A C A T T G T G A G C C A A A A T A A T A A C A A A G C C A G G T T T T C A G G C T G A C G G C T G T G A A T C T G A A A C T C T A G G G G A G C C T T T T T A A T T T G T T T T T C T T G A G A C A G G G T A T C T CT GTG TAACC C TGG C TATC C TGGAAC TCAC TC T A T A G A C C A G G C T G G C C T C G A A C T C A C A G A T A T C T G T C TGC C T C TGC C TC C T A A G T A C T G G G A T T C A A G G C A T G T A C G G C A A C T G C C C A G CTAAAA T A T T A T T T A T A A C A T C C A C TTTC T G G G T T T T T T G T T T T T A A A A C A T A C T T T T T T TT T T A A C A C T G G G C C A T T T C TAACA T T T C T G C A C A G A A G T G G A T T T A G C T C A G A T T A A C T TAATT TTGAAAAGGTAACAGTAC TGTTTTTTTTTCC TTAATGCTCTTATGAAAACAATGT TGAAT TTACAGAGGGC TTTTTGTGTTTTTGTTTTTTTGTTTT TTGTTTTTTGTTTGGGAG C T G G G G A C C G A A C C C A G G G C C T T G A G C T T C C T A G G T A A G C G C TC TAT CAC T G A G C T A A A T C C C CAGC C G A C A G A G G G C T T T T T T A G T A G T G T G T A G T G A G T A T C AGC T G A T T C G A G C T A A TAACTTTACCTTGGGGTTTTTGTTTGTTTGTTTTCCTGGTCTCCTTTGCCTGACCTCTTT T TAAATTATGTGTAATTCAAA GACTATTCAAGTGATGG TTAGTCATGAGTCGTGACGTT T G A C T G G T G T G A A G T A A A TTC T T G T T C T T A A G A A A A A A A A A A

2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000

Fig. 1. Nucleotide sequence and deduced amino acid sequence of rat PepT1 cDNA. Nucleotide numbers are indicated at the start and end of each line.

K,-i. Miyamoto et al. / Biochimica et Biophysica Acta 1305 (1996) 34-38

36

I - - - - - ~ M1-----------------4~

F---"--

Rat

1 :M G M S K S R G C F G Y P L S

Rabbit

1: . . . . . . L S . . . . . . . . . . . . . . . . . . . . . .

I F~FF II.V.~VF~C. F C r R .FSYY T R A L L V L Y F R N F L G W D D D L S T ~ I Y H T F V

h~an

1 ....... HSP ....... --....._-I.-__-_.I.. ,I.I...T..IS...N

--M2

I

I . . . . . . I .... N . . . V

1

M3-------I

Rat

6 1 :A L C Y L T P

Rabbit

61: ...............

human

61: .....................................

I LGALIADSWLGKFKT

F-

IVSLS IVYT IGQAVI SVSS INDLTDHDHDGSPNNL

A ......... W ...........

M4--1

......

..........

pL

T.L..V.E...biN...T.DS..V T ...........

N...T.DS..V

I

M5

Rat

1 2 1 :H V A L S M

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1 2 1 : . . . V C . . . . L ...l.-.....,.-...r. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

human

1 2 1 : . . V . . L . . . . . .I. • • • • .I.

Rat

1 8 1 :T P I L R V Q Q C G I H S Q Q A C Y

Rabbit

1 8 1 : . . M V . . . . . . . . V K . . . . . . . . . I . . Z .... S . . . . I I . . . . . . . . K . . . . . L S . . V . . .

human

181: . .M .......... •, **,*****

Rat

2 4 1 :R F A I K N R F R H R S K A F

Rabbit

241:C

............

human

241:G

........................................

Rat

3 0 1 :G S R W T L Q A T T M T G K

Rabbit

301: ...........

S.R..IL

human

301: ........... • **********

S .... A L . . . . . . . . . . . . . . . . . . . . . F, . .L . . . . . . . . . . . . . . . . , ** *********** *** , *** *** ******** *******

I G L A L ~

KPCVSAFGGDQFEEGQEKQRNRFFS

I

I FYLAZNAGSLLSTZ

M6------~

PLAFGVPAALMAVAL

~VFVLGSGMYKKFQPQGNIMGKVAKC

K ................................ ********* ** **** **** ********

I

K ............. ***** ** ,,,

~------- M 7

L

PKREHWLDWAKEKYDERL

I

IGTI E I QPDQMQTVNAI ...........

I

I SQIKMVTKVMFLYI

Q .... A . . . . . . . . . . . . . . .

PLPMFWALFDQQ

A ...... R.L ................ R ..................

M8

I

LIVI MVPIVDAVVYPL

T.. ,I.L...M

IAKCGFNF%~KK

............

L .......

M9-------~ LAS MAFVVAA

I

v

Rat

3 6 1 :b ~ V G M F

Rabbit

3 6 1 : . .I . . . . . . . . . .

IVQVE I DKTL PVF PSGNQVQ I KVLN IGNNDMAVYF

human

361: .A...V ........................

Rat

4 2 1 :M S Q T D T F M T F D V D Q L T S

Rabbit

421: .... NE .... NE.T ..... IT.-..Q-..MITPSL.A.Q

. . . . . . . A . . N .... N . . . T .

human

4 2 1 : .... N A . . . . . . N K . . R . . I

...... -..A.TDD.KQ.Q

....... A..H.Q

Rat

4 8 1 :K P E N G E N G Z R F V S T L N E M I T

I KMSGK~E~VTS~SASNYQFF

Rabbit

479: .SD ......... N.YSQP.NVT

....... RIA.YN..E

.... T..V.GF,VSSAG.SEQ.

human

480: ............

...... A.IS.YN..T

....... I.GF..SS...P.Q.

Rat

5 4 1 :S S D F K S S N L D F G S A Y T Y V I

Rabbit

539:RR..E.PY.E

human

540 :QPN.NTFY,

Rat

501 :SVTGLEF SYSQAPSNMKSVLQAGWLLTVA

Rabbit

5 9 8 : .I . . . . . . . . . . . . . . . . . . . . . . . . . . .

V ......... G..QIN

. . . . . . I . . . A ....

hLunan

599: .............................

V ......... G..Q.S

. . . . . . I . . . A ....

Rat

6 6 1 :V C I I F A I M A R F Y T Y

INPAEI EAQFDEDEKKKGVGKENPYSSL

Rabbit

658: . .v ...........

V ......... E ...... NPE.NDL.P..A-.

human

659: . .V ...........................

A...L

............

PGKNVTVAQ

KA.E ....... V.SEN.IISL..QT..LN. K..E ...........

T.NISL..EM..LGP

V INVSS PGS PGVTTVAHEFEPGHRRTLLVWGPNLYRVVKDGLNQ

N.F..L...T

RSRASDGCLEWKEFED

....... L.T.Q.TG-.PQ,T

PSGQ~DYT

I pPNTVNMALQ

......... M...W

........

V V INTTE IAPNC

MIO-

I

I pQYFLLTCGEVVF

....... I.S .....

E . . . . . . . I V Q - . K N . S . P . . . V .... S A . . . . . . . . . . . . . . . . . . . . . .

- - I

I

M11 - - - - - - - I

I

IGNI IVL IVAEAGRFDKQWAEYVLFAS

NRLE.S...

M12LLLV

- EPVSQTNM .... Q .

-FMSGAN..KQ.

Fig. 2. Comparison of amino acid sequences of rat, rabbit and human PepT1. Amino acids are indicated by single amino acids letter abbreviations. Results identical to those in rat PepT1 are indicated by dots, with conserved in all three sequences indicated by asterisks. Hyphens represent gaps introduced to optimum alignment. The 12 predicted transmembrane regions (M1 to M12) are underlined. Two boxed sequences correspond to the consensus sequences (ERFSYYG and ALGTGG) present in other nonmammalian transporters. Residue numbers are shown on the left. The V above letter represents glycosylation sites. The circled S represents PKC site and the boxed T represents PKA site.

reveals the presence of 12 putative transmembrane domains with a long (204 amino acids) hydrophilic segment between transmembrane domains 9 and 10 (Fig. 2). This hydrophilic segment contains five putative N-linked glycosylation sites. When modeled to accommodate all the transmembrane domains with the long hydrophilic loop on the extracellular side, the predicted protein contains potential phosphorylation sites for protein kinase C (PKC) (Ser357) and protein kinase A (PKA) (Thr-362). Rabbit PepT1 zlso contains corresponding phosphorylation sites for each of these kinase [5]. Recent studies of the H÷/oligopeptide transporter in the human cell line CaCo-2 revealed that activation of PKC in these cells decreases the maximal

transport rate (Wmax) of the transporter but has no affect on apparent substrate affinity (K m) [11]. The single PKC phosphorylation site of rabbit PepT1 is conserved in human and rat proteins, and HPepT1 have an additional putative PKC site close to the COOH-terminus. The PKA site of rabbit and rat PepT1, however, is not conserved in the human protein. As shown in Fig. 2, comparison of the amino acid sequence of rat PepT1 with those of the rabbit and human proteins reveals a high degree of homology (77% and 83% identity, respectively) [12]. Amino acids in the membranespanning regions are especially well conserved among the three species. Sequence differences among the three proteins are apparent in the loops connecting the 12 predicted transmembrane domains, especially in the large extracellular loop, as well as in the putative intracellular COOHterminal sequence. The PepTl amino acid sequence does not show significant homology to other known mammalian sequences. However, Liang et al. [6] reported that HPepT1 shows weak homology (25% identity) to the H÷-dependent nitrate transporter CHL1 from plants (Arabidopsis thaliana) [13] and a peptide permease from Saccharomyces cereuisiae [14]. Furthermore, a stretch of seven residues (ERFSYYG) of HPepT1 is conserved in the yeast peptide permease but not in the nitrate transporter [6], and another stretch of six amino acids (ALGTGG) is conserved in the plant nitrate transporter but not in the yeast permease [6]. As shown in Fig. 2, the conservation of these motifs in rat, human and rabbit PepTl suggests that they may play important structural or functional roles. Of the various rat tissues examined, a 3.0-kb rat PepT1 mRNA was detected in kidney and small intestine (Fig. 3). The strong hybridization signal in duodenum jejunum, and ileum is consistent with these segments being the principal

PepT1 ~3.0 kb

Fig. 3. Tissue distribution of rat PepT1 mRNA. Total RNA (15/zg) from each tissue was resolved on a 1.2% agarose gel containing formaldehyde, transferred to a nylon membrane, and hybridized with a 32 P-labeled rat PepT1 cDNA probe. The positions of 28S and 18S rRNA and the ~ 3.0-kb PepT1 mRNA are indicated.

K.-i. M(vamoto et al. / Biochimica et Biophysica Acta 1305 (1996) 34-38

site of intestinal absorption of protein digestion products. In the kidney, peptide transporters serve to reabsorb filtered peptides, peptide-derived antibiotics, and peptides produced as a result of the action of luminal peptidase [15]. Recently, Liu et al. [16] isolated a peptide transporter, PepT2, from human kidney that is ~ 50% identical to PepTl [16]. Multiple peptide transporters are therefore present in the kidney. Although rabbit PepT1 mRNA is present in brain and liver, rat PepT1 mRNA was not detected in these tissues. The functional characteristics of the H+/peptide cotransporter have been investigated in detail only in intestine and kidney. The functions of peptide transporters in other tissues remain unknown. A putative peptide transporter, HPT-1, has also been isolated with a monoclonal antibody that blocked cephalexin uptake into CaCo-2 cells [17]. However, recent data suggest that HPT-1 is not the actual H+-coupled oligopeptide transporter but rather acts as a modulator of endogenous oligopeptide transport in CaCo-2 cells [12]. In contrast, microinjection of cRNA of rat PepT1 synthesized by in vitro transcription into oocytes induced a pH dependent transport activities of a dipeptide, [HC]glycylsarcosine as well as /3-1actam antibiotics, ceftibuten and cefadroxil. Uptake of /3-1actam antibiotics by oocytes injected with rat intestinal total mRNA was almost completely abolished by an antisense oligonucleotide against rat PepT1 (I. Tamai et al., unpublished work). These observations suggest that rat PepT1 has a major role of the intestinal absorption of oligopeptides and /3-1actam antibiotics. Next, we examined the changes in expressions of PepTl gene in rat jejunum during development. As shown in Fig. 4, the level of intestinal PepTl mRNA was dramatically increased in 10-day-old rats, and then decreased reaching the adult level by day 28 after birth. When an animal develops, the developing gut faces major changes in functional demands placed on it [18]. First, at birth, the gut replaces the placenta as the site of nutrient acquisition. Second, at the time of weaning, animals make major qualitative changes in diet and, in the case of rats, often replace a high-protein milk diet with an adult diet typically containing more carbohydrate than protein. Third, growing animals require quantitatively more nutrients. Intestinal PepT1 mRNA was most highly expressed at birth, suggesting that it is important to absorb the peptides derived from a high-protein milk diet. On the other hand, amino acid absorption in rat small intestine typically declines around two fold from birth to adolescence [19,20]. The main mechanism underlying the decline in uptake rate is a decrease in Vmax and not changes in K,, brush border membrane permeability, or electrochemical sodium gradient [19,20]. Second, the postnatal decline is typically greater for essential amino acids than for nonessential ones because neonatal animals have a much larger requirement for essential amino acids to synthesize new proteins required for fast growth [20]. The level of intestinal PepT1 mRNA in 4-old-days increased about

A)

37

Days 2 4 14 21 28 70

PepT1 GAPDH 4ooI

or'

0 10 20 30 40 50 60 70

Days Fig. 4. (A) Northern blot analysis of PepT1 mRNA in the jejunum from developing rats. Total cellular RNA was isolated from pooled tissues. Each lane contains ~ 10 /xg of total RNA from the jejunum of developing rats. Hybridization with a probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed to normalize the data for differences in RNA loading. (B) Relative intensities of PepT1 mRNAs in developing rats. Data were quantitated by image analysis with a Fuji BAS 2000 system. The levels of each mRNA are expressed as percentages of those in 2-day-old rats.

3.6-fold in that of adult rats, suggesting that intestinal peptide transport system is more important for neonatal animals. The postnatal development of the rat small intestine is characterized by changes in a variety of enzyme and transporter during the third week of life [8,21]. There is strong evidence that these changes are induced by dietary change from milk to solid food and by hormonal regulation (glucocorticoid and thyroid hormones) [21]. Further studies are need to clarify the regulation of PepT1 in developing rats.

References [1] Ganapathy, V. and Leibach, F.H. (1985) Am. J. Physiol. 249, G153-G160. [2] Ganapathy, V. and Leibach, F.H. (1991) Curt. Opin. Cell Biol. 3, 695 -701. [3] Tsuji, A. (1995) Intestinal absorption of /3-1actam antibiotics in

38

[4] [5]

[6]

[7] [8]

[9]

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K.-i. Miyamoto et al. / Biochimica et Biophysica Acta 1305 (1996) 34-38

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