David T. Thwaites

Substrate upregulation of the human small intestinal peptide transporter, hPepT1

1 Molecular mechanisms underlying physiological adaptation to increased levels of dietary peptides have been elucidated by studying the response to the substrate glycyl‐L‐glutamine (Gly‐Gln) of the proton‐coupled peptide transporter, hPepT1, in the Caco‐2 human intestinal cell line. Vmax for apical uptake of [14C]glycyl‐[14C]sarcosine was increased 1.64 (± 0.34)‐fold after incubation of Caco‐2 cells for 3 days in a peptide‐rich medium (4 mM Gly‐Gln replacing 4 mM Gln). 2 A full‐length Caco‐2 hPepT1 cDNA clone was identical to human small intestinal hPepT1 with the exception of a single amino acid substitution Ile‐662 to Val. Transcript sizes, on Northern blots of Caco‐2 poly(A)+ RNA probed with a 630 bp 5′ hPepT1 cDNA probe, correspond to the reported band pattern seen with human small intestinal RNA. The dipeptide‐induced increase in substrate transport was accompanied by a parallel increase of 1.92 (± 0.30)‐fold (n= 9) in hPepT1 mRNA. This was in part due to an increase in hPepT1 mRNA half‐life from 8.9 ± 1.1 to 12.5 ± 1.6 h (n= 3), but the increase in half‐life does not account fully for the observed increase in mRNA levels, suggesting that there was also a dipeptide‐mediated increase in hPepT1 transcription. 3 Anti‐hPepT1‐specific antipeptide antibodies localized hPepT1 exclusively to the apical membrane of human small intestinal enterocytes and Caco‐2 cells. Gly‐Gln supplementation of media resulted in a 1.72 (± 0.26)‐fold (n= 5) increase in staining intensity of Caco‐2 cells. 4 We conclude that Caco‐2 cells provide an appropriate model for the study of adaptation of intestinal hPepT1, at the molecular level, to increased levels of dietary peptide. The magnitude of functional increase in apical peptide transport activity in response to Gly‐Gln can be fully accounted for by the increased levels of hPepT1 protein and mRNA, the latter mediated by both enhanced hPepT1 mRNA stability and increased transcription. The signalling pathway between increased dietary peptide and hPepT1 upregulation, therefore, involves direct action on the enterocyte, independent of hormonal and/or neural control.

H+-coupled nutrient, micronutrient and drug transporters in the mammalian small intestine.

The H+‐electrochemical gradient was originally considered as a driving force for solute transport only across cellular membranes of bacteria, plants and yeast. However, in the mammalian small intestine, a H+‐electrochemical gradient is present at the epithelial brush‐border membrane in the form of an acid microclimate. Over recent years, a large number of H+‐coupled cotransport mechanisms have been identified at the luminal membrane of the mammalian small intestine. These transporters are responsible for the initial stage in absorption of a remarkable variety of essential and non‐essential nutrients and micronutrients, including protein digestion products (di/tripeptides and amino acids), vitamins, short‐chain fatty acids and divalent metal ions. Proton‐coupled cotransporters expressed at the mammalian small intestinal brush‐border membrane include: the di/tripeptide transporter PepT1 (SLC15A1); the proton‐coupled amino‐acid transporter PAT1 (SLC36A1); the divalent metal transporter DMT1 (SLC11A2); the organic anion transporting polypeptide OATP2B1 (SLC02B1); the monocarboxylate transporter MCT1 (SLC16A1); the proton‐coupled folate transporter PCFT (SLC46A1); the sodium–glucose linked cotransporter SGLT1 (SLC5A1); and the excitatory amino acid carrier EAAC1 (SLC1A1). Emerging research demonstrates that the optimal intestinal absorptive capacity of certain H+‐coupled cotransporters (PepT1 and PAT1) is dependent upon function of the brush‐border Na+–H+ exchanger NHE3 (SLC9A3). The high oral bioavailability of a large number of pharmaceutical compounds results, in part, from absorptive transport via the same H+‐coupled cotransporters. Drugs undergoing H+‐coupled cotransport across the intestinal brush‐border membrane include those used to treat bacterial infections, hypercholesterolaemia, hypertension, hyperglycaemia, viral infections, allergies, epilepsy, schizophrenia, rheumatoid arthritis and cancer.

Structure, function and immunolocalization of a proton‐coupled amino acid transporter (hPAT1) in the human intestinal cell line Caco‐2

The human orthologue of the H+‐coupled amino acid transporter (hPAT1) was cloned from the human intestinal cell line Caco‐2 and its functional characteristics evaluated in a mammalian cell heterologous expression system. The cloned hPAT1 consists of 476 amino acids and exhibits 85 % identity with rat PAT1. Among the various human tissues examined by Northern blot, PAT1 mRNA was expressed most predominantly in the intestinal tract. When expressed heterologously in mammalian cells, hPAT1 mediated the transport of α‐(methylamino)isobutyric acid (MeAIB). The cDNA‐induced transport was Na+‐independent, but was energized by an inwardly directed H+ gradient. hPAT1 interacted with glycine, l‐alanine, l‐proline, α‐aminoisobutyrate (AIB) and γ‐aminobutyrate (GABA), as evidenced from direct transport measurements and from competition experiments with MeAIB as a transport substrate. hPAT1 also recognized the d‐isomers of alanine and proline. With serine and cysteine, though the l‐isomers did not interact with hPAT1 to any significant extent, the corresponding d‐isomers were recognized as substrates. With proline and alanine, the affinity was similar for l‐ and d‐isomers. However, with cysteine and serine, the d‐isomers showed 6‐ to 8‐fold higher affinity for hPAT1 than the corresponding l‐isomers. These functional characteristics of hPAT1 closely resemble those that have been described previously for the H+‐coupled amino acid transport system in Caco‐2 cells. Furthermore, there was a high degree of correlation (r2= 0.93) between the relative potencies of various amino acids to inhibit the H+‐coupled MeAIB transport measured with native Caco‐2 cells and with hPAT1 in the heterologous expression system. Immunolocalization studies showed that PAT1 was expressed exclusively in the apical membrane of Caco‐2 cells. These data suggest that hPAT1 is responsible for the H+‐coupled amino acid transport expressed in the apical membrane of Caco‐2 cells.

H+/di-tripeptide transporter (PepT1) expression in the rabbit intestine

In order to examine the intestinal expression of the recently cloned H+/di-tripeptide transporter (PepT1), oligonucleotide probes were synthesized and their specificity confirmed by Northern blot analysis of rabbit jejunal RNA. In situ hybridization studies, using these probes, show that PepT1 is expressed all along the small intestine and at a very much reduced level in the colon. In contrast, PepT1 mRNA was not detected in the stomach, sacculus rotundus or caecum. Microscopic examination of tissue sections showed PepT1 expression to be restricted to intestinal epithelium with no detectable expression in the lamina propria, muscularis mucosae, muscularis or serosa. The accumulation of PepT1 mRNA along the crypt-villus axis was also investigated. In all regions of the small intestine (in duodenum, jejunum and ileum), PepT1 mRNA was undetectable in deeper epithelial cells of the crypts. Expression was first detectable at or near the crypt-villus junction, the amount of PepT1 mRNA increasing rapidly in the lower villus to a maximum approximately 100–200 μm from this point. Along the length of the small intestine PepT1 mRNA was most abundant in duodenal and jejunal enterocytes, with lower levels in the ileal epithelium. PepT1 expression is greatly depressed in the follicle-associated epithelium of the Peyers patch relative to both interfollicular and adjacent “normal” villi. These data are discussed in the context of the known physiological role of PepT1 in the gastrointestinal tract.

Optimal absorptive transport of the dipeptide glycylsarcosine is dependent on functional Na+/H+ exchange activity.

Abstract. Optimal nutrient absorption across the intestinal epithelium is dependent on the co-ordinated activity of a number of membrane transporters. Di/tripeptide transport across the luminal membrane of the intestinal enterocyte is mediated by the H+-coupled di/tripeptide transporter hPepT1. hPepT1 function is dependent on the existence of a pH gradient (maintained, in part, by the action of the Na+/H+ exchanger NHE3) across the apical membrane of the small intestinal epithelium. The physiological problem addressed here was to determine how two transporters (hPepT1 and NHE3), involved in nutrient absorption and pHi homeostasis, function co-operatively to maximise dipeptide absorption when both operate sub-optimally at typical mucosal surface pH values (pH 6.1–6.8). Functional hPepT1 activity in human intestinal epithelial (Caco-2) cell monolayers was determined by measurement of apical uptake and apical-to-basolateral transport of the dipeptide glycylsarcosine. The dependence of hPepT1 on NHE3 activity was measured (either after Na+ removal or addition of the NHE3-selective inhibitor S1611) using both Caco-2 cell monolayers and hPepT1-expressing Xenopus laevis oocytes. Apical glycylsarcosine uptake in Caco-2 cell monolayers was modulated by apical pH, extracellular Na+, incubation time and S1611. Uptake in hPepT1-expressing oocytes was independent of Na+ or S1611. We conclude that functional NHE3 activity is required to allow optimal absorption of dipeptides across the human intestinal epithelium.

Stereoselective uptake of β-lactam antibiotics by the intestinal peptide transporter

1 The stereoselective transport of β‐lactam antibiotics has been investigated in the human intestinal epithelial cell line, Caco‐2, by use of D‐ and L‐enantiomers of cephalexin and loracarbef as substrates. 2 The L‐isomers of cephalexin, loracarbef and dipeptides displayed a higher affinity for the oligopeptide/H+‐symporter in Caco‐2 cells than the D‐isomers. This was demonstrated by inhibition of the influx of the β‐lactam, [3H]‐cefadroxil. 3 By measurement of the substrate‐induced intracellular acidification in Caco‐2 cells loaded with the pH‐sensitive fluorescent dye BCECF (2′,7′‐bis(2‐carboxyethyl)‐5‐(6)‐carboxy‐fluorescein), it was demonstrated for the first time that L‐isomers of β‐lactams not only bind to the peptide transporter with high affinity but are indeed transported. 4 Efficient proton‐coupled transport of L‐β‐lactam antibiotics was also shown to occur in Xenopus laevis oocytes expressing the cloned peptide transporter PepTl from rabbit small intestine. 5 Both cell systems therefore express a stereoselective transport pathway for β‐lactam antibiotics with very similar characteristics and may prove useful for screening rapidly the oral availability of peptide‐derived drugs.

Angiotensin‐converting enzyme (ACE) inhibitor transport in human intestinal epithelial (Caco‐2) cells

1 The role of proton‐linked solute transport in the absorption of the angiotensin‐converting enzyme (ACE) inhibitors captopril, enalapril maleate and lisinopril has been investigated in human intestinal epithelial (Caco‐2) cell monolayers. 2 In Caco‐2 cell monolayers the transepithelial apical‐to‐basal transport and intracellular accumulation (across the apical membrane) of the hydrolysis‐resistant dipeptide, glycylsarcosine (Gly‐Sar), were stimulated by acidification (pH 6.0) of the apical environment. In contrast, transport and intracellular accumulation of the angiotensin‐converting enzyme (ACE) inhibitor, lisinopril, were low (lower than the paracellular marker mannitol) and were not stimulated by apical acidification. Furthermore, [14C]‐lisinopril transport showed little reduction when excess unlabelled lisinopril (20 mm) was added. 3 pH‐dependent [14C]‐Gly‐Sar transport was inhibited by the orally‐active ACE inhibitors, enalapril maleate and captopril (both at 20 mm). Lisinopril (20 mm) had a relatively small inhibitory effect on [14C]‐Gly‐Sar transport. pH‐dependent [3H]‐proline transport was not inhibited by captopril, enalapril maleate or lisinopril. 4 Experiments with BCECF[2′,7′,‐bis(2‐carboxyethyl)‐5(6)‐carboxyfluorescein]‐loaded Caco‐2 cells demonstrate that dipeptide transport across the apical membrane is associated with proton flow into the cell. The dipeptide, carnosine (β‐alanyl‐l‐histidine) and the ACE inhibitors, enalapril maleate and captopril, all lowered intracellular pH when perfused at the apical surface of Caco‐2 cell monolayers. However, lisinopril was without effect. 5 The effects of enalapril maleate and captopril on [14C]‐Gly‐Sar transport and pHi suggest that these two ACE inhibitors share the H+‐coupled mechanism involved in dipeptide transport. The absence of pH‐dependent [14C]‐lisinopril transport, the relatively small inhibitory effect on [14C]‐Gly‐Sar transport, and the absence of lisinopril‐induced pHi changes, all suggest that lisinopril is a poor substrate for the di/tripeptide carrier in Caco‐2 cells. These observations are consistent with the greater oral availability and time‐dependent absorption profile of enalapril maleate and captopril, compared to lisinopril.

The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers

We determined the extent of Na+-independent, proton-driven amino acid transport in human intestinal epithelia (Caco-2). In Na+-free conditions, acidification of the apical medium (apical pH 6.0, basolateral pH 7.4) is associated with a saturable net absorption of glycine. With Na+-free media and apical pH set at 6.0, (basolateral pH 7.4), competition studies with glycine indicate that proline, hydroxyproline, sarcosine, betaine, taurine, β-alanine, α-aminoisobutyric acid (AIB), α-methylaminoisobutyric acid (MeAIB), τ-amino-n-butyric acid and l-alanine are likely substrates for pH-dependent transport in the brush border of Caco-2 cells. Both d-serine and d-alanine were also substrates. In contrast leucine, isoleucine, valine, phenylalanine, methionine, threonine, cysteine, asparagine, glutamine, histidine, arginine, lysine, glutamate and d-aspartate were not effective substrates. Perfusion of those amino acids capable of inhibition of acid-stimulated net glycine transport at the brush-border surface of Caco-2 cell monolayers loaded with the pH-sensitive dye 2′,7′-bis(2-carboxyethyl-5(6)-carboxyfluorescein) (BCECF) caused cytosolic acidification consistent with proton/amino acid symport. In addition, these amino acids stimulate an inward short-circuit current (Isc) in voltage-clamped Caco-2 cell monolayers in Na+-free media (pH 6.0). Other amino acids such as leucine, isoleucine, phenylalanine, tryptophan, methionine, valine, serine, glutamine, asparagine, d-aspartic acid, glutamic acid, cysteine, lysine, arginine and histidine were without effect on both pHi and inward Isc. In conclusion, Caco-2 cells express a Na+-independent, H+-coupled, rheogenic amino acid transporter at the apical brush-border membrane which plays an important role in the transepithelial transport of a range of amino acids across this human intestinal epithelium.

The SLC36 family of proton‐coupled amino acid transporters and their potential role in drug transport

Members of the solute carrier (SLC) 36 family are involved in transmembrane movement of amino acids and derivatives. SLC36 consists of four members. SLC36A1 and SLC36A2 both function as H+‐coupled amino acid symporters. SLC36A1 is expressed at the luminal surface of the small intestine but is also commonly found in lysosomes in many cell types (including neurones), suggesting that it is a multipurpose carrier with distinct roles in different cells including absorption in the small intestine and as an efflux pathway following intralysosomal protein breakdown. SLC36A1 has a relatively low affinity (Km 1–10 mM) for its substrates, which include zwitterionic amino and imino acids, heterocyclic amino acids and amino acid‐based drugs and derivatives used experimentally and/or clinically to treat epilepsy, schizophrenia, bacterial infections, hyperglycaemia and cancer. SLC36A2 is expressed at the apical surface of the human renal proximal tubule where it functions in the reabsorption of glycine, proline and hydroxyproline. SLC36A2 also transports amino acid derivatives but has a narrower substrate selectivity and higher affinity (Km 0.1–0.7 mM) than SLC36A1. Mutations in SLC36A2 lead to hyperglycinuria and iminoglycinuria. SLC36A3 is expressed only in testes and is an orphan transporter with no known function. SLC36A4 is widely distributed at the mRNA level and is a high‐affinity (Km 2–3 µM) transporter for proline and tryptophan. We have much to learn about this family of transporters, but from current knowledge, it seems likely that their function will influence the pharmacokinetic profiles of amino acid‐based drugs by mediating transport in both the small intestine and kidney.

H+/solute-induced intracellular acidification leads to selective activation of apical Na+/H+ exchange in human intestinal epithelial cells

The intestinal absorption of many nutrients and drug molecules is mediated by ion-driven transport mechanisms in the intestinal enterocyte plasma membrane. Clearly, the establishment and maintenance of the driving forces - transepithelial ion gradients - are vital for maximum nutrient absorption. The purpose of this study was to determine the nature of intracellular pH (pH(i)) regulation in response to H(+)-coupled transport at the apical membrane of human intestinal epithelial Caco-2 cells. Using isoform-specific primers, mRNA transcripts of the Na(+)/H(+) exchangers NHE1, NHE2, and NHE3 were detected by RT-PCR, and identities were confirmed by sequencing. The functional profile of Na(+)/H(+) exchange was determined by a combination of pH(i), (22)Na(+) influx, and EIPA inhibition experiments. Functional NHE1 and NHE3 activities were identified at the basolateral and apical membranes, respectively. H(+)/solute-induced acidification (using glycylsarcosine or beta-alanine) led to Na(+)-dependent, EIPA-inhibitable pH(i) recovery or EIPA-inhibitable (22)Na(+) influx at the apical membrane only. Selective activation of apical (but not basolateral) Na(+)/H(+) exchange by H(+)/solute cotransport demonstrates that coordinated activity of H(+)/solute symport with apical Na(+)/H(+) exchange optimizes the efficient absorption of nutrients and Na(+), while maintaining pH(i) and the ion gradients involved in driving transport.