The Peptide Histidine Transporters

Thyroid Factor

The Natural Thyroid Diet

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Little information is known concerning the function of the PHT transporters. The rat peptide/histidine transporters (rPHT1 and rPHT2) have both shown high affinity for histidine and the ability to transport dipeptides (Yamashita et al., 1997; Sakata et al., 2001). Rat PHT1 expression in Xenopus oocytes revealed a high-affinity, proton-dependent histidine uptake that was inhibited by several di- and tripeptides, but not other amino acids. Rat PHT1 also shows high affinity for the p-ala-his dipeptide, carnosine (Yamashita et al., 1997). Rat PHT2 reconstitution in liposomes modeling the lysosomal environment showed proton-dependent transport activity with histidine and dipeptides, but not with amino acids (Sakata et al., 2001). However, studies in our laboratory suggest that hPHT1

Table 7.5. Therapeutic compounds shown to interact with PepTl

Substrates Inhibitors

Cephalosporins

Cefaclor

Cefadroxil

Cefamandole

Cefatrizine

Cefepine

Cefixime

Cefodizime

Cefpirome

Cefroxadine

Ceftibuten

Cefotaxime

Ceftriaxone

Cephalexin

Cephradine

Loracarbef

Moxalactam

Latamoxef

Penicillins Benzylpenicillin

Cloxacillin

Cyclacillin

Dicloxacillin

Metampicillin

Oxacillin

Phenoxymethyl-penicillin Propicillin

ACE inhibitors

Benazepril

Captopril

Enalapril

Fosinopril

Quinalapril

Antivirals

Valaciclovir

Valganciclovir

Others Bestatin a-methyldopa-phenylalanine Pro-Phe-alendronate Renin inhibitors Thrombin inhibitors Thyrotropin-releasing hormone

Carbenicillin

Benazeprilat Enalaprilat Fosinoprilat Quinalaprilat

Glibenclamide Nateglinide Lys[Z(NO2 )]-Pro mediates the transport of not only carnosine and L-histidine, but also valacyclovir in a proton-dependent, sodium-independent manner (Bhardwaj et al., 2005a). Nevertheless, there is still much to elucidate concerning the affinity and substrate specificity of these peptide transporters.

7.3.1.3 The Regulation of Peptide Transporters

Information concerning the regulation mechanisms of peptide transporters is limited to PepTl. Earlier studies have shown that PepTl transport activity changes as a response to diet regimens (Thamotharan et al., 1998, 1999a; Walker et al., 1998; Shiraga et al., 1999; Ihara et al., 2000, Adibi, 2003). PepTl mRNA expression levels significantly increased in Caco-2 cells cultured in a dipeptide supplemented medium (Thamotharan et al., 1998; Walker et al., 1998). Other studies have demonstrated that malnourishment upregulates PepT1 expression in rat intestines (Ogihara et al., 1999, Thamotharan et al., 1999a; Ihara et al., 2000), suggestive of transcriptional regulation.

Shiraga et al. (1999) performed a characterization of the rPepT1 promoter to evaluate how factors including the diet participate in the transcriptional activation of the rPepT1 gene. They suggested that the AP-1 binding site (TGACTCAG, nt -295) and the AARE-like element-binding site (CATGGTG, nt -277) regions were associated with dietary protein content regulation of rPepT1. Deletion analysis of the hPEPT1 promoter region in Caco-2 cells suggested that the region spanning —172 to -35 bp was essential for basal transcriptional activity, demonstrating the significant role of the Sp1 nuclear transcription factor in the basal transcriptional regulation of hPepT1 (Shimakura et al., 2005).

The regulatory effects of secondary messengers on PepT activity are still controversial. Activation of PKC is suggested to be responsible for the downregulation of Gly-Sar uptake into Caco-2 cells (Brandsch et al., 1994). Other investigators have also illustrated that transepithelial peptide transport is inhibited by PKC activation, or by an increase in intracellular Ca2+ (Wenzel et al., 1999, 2002). Additionally, di- and tripeptide transport activity has been modified by agents that interfere with intracellular cAMP levels (Muller et al., 1996; Berlioz et al., 1999, 2000).

The effect of hormones on peptide transport has not been comprehensively analyzed; however, several summary reviews are available (Meredith and Boyd, 2000; Gangopadhyay et al., 2002; Adibi, 2003). Several studies have demonstrated an effect of insulin (Thamotharan et al., 1999b) and leptin (Buyse et al., 2001) on PepT1 activity in Caco-2 cells. Dipeptide uptake into Caco-2 cells is stimulated by insulin (5 nM) treatment, with an observed apparent increase in the transporter capacity (Vmax increased twofold) with no alteration in the Km and hPepT1 mRNA levels (Thamotharan et al., 1999b). These studies suggest that the effect in transport capacity was due to an increase in the insertion of PepT1 protein in the plasma membrane from a preformed cytoplasmic pool. Similar observations were obtained in Caco-2 cells treated with leptin (Buyse et al., 2001).

Ashida et al. (2002) reported the effect of thyroid hormone 3, 5, 3'-L-triiodo-thyronine (T3) on the expression and transport activity of PepT1 in Caco-2 cells suggesting that the changes in dipeptide uptake were associated with the inhibition of the transcription of PepT1 mRNA and/or with a change in the mRNA stability, but the precise mechanism of the T3 effect on [14C]glycylsarcosine transport was not clearly elucidated. Furthermore, in vivo studies in euthyroid and hyperthyroid rats have demonstrated the effect of thyroid hormone on the activity and expression of PEPT1 in the small intestine (Ashida et al., 2004). The [14C]glycylsarcosine uptake by everted small intestinal preparations was significantly decreased in hyperthyroid rat. The mean portal vein concentrations after intrajejunal administration of [14C]glycylsarcosine were also decreased in hyperthyroid rats. Moreover, hyperthyroidism caused a significant decrease in the expression of PEPT1 mRNA and protein in the small intestine. These results show the relevancy of hormonal regulation in the expression and activity of peptide transporters, providing useful information for protein nutrition and drug treatment in patients with hyperthyroidism.

Several studies have demonstrated the capability of the small intestine to compensate for possible nutritional deficiencies caused by tissue injury or resection (Tanaka et al., 1998; Merlin et al., 1998, 2001; Takahashi et al., 2001; Ziegler et al., 2002). Studies performed in sections of Short-bowel syndrome patients have revealed that hPepT1 mRNA and protein expression were up regulated in the colon mucosa, suggesting hPepT1 expression maybe an adaptation process in response to gut mucosal damage (Ziegler et al., 2002). Furthermore, Merlin et al. (1998, 2001) have suggested that the aberrant PepT1 expression under chronic disease states implicates PepT1 function in intestinal inflammatory processes.

It has been recognized that genetic polymorphism of membrane transporters may affect their function in different ways. SNPs localized on exons might modify the intrinsic activity by changing the affinity to substrates (Km) and/or the protein translocation ability or capacity (Vmax). Furthermore, the capacity of the transporter can also be altered by changes in the protein expression level or impaired subcellular sorting of the protein to appropriate domains of the plasma membrane (Ishikawa et al., 2004).

Peptide transporters polymorphisms have been scarcely studied. The only functional report testing polymorphisms and their impact on the transport of substrates was recently published by Zhang et al. (2004). In this study, nine nonsynonymous SNPs were identified in a population of 44 individuals of different ethnicities. The hPepT1 wild-type sequence as well as the hPepT1 sequences containing the individual SNPs were amplified and transiently transfected into HeLa cells, where the transport kinetics of [14C]Gly-Sar was analyzed. Western blot and immunocyto-chemical analyses were performed to establish the amount and expression of the hPepT1 protein in the cells. Their results showed that only the nonsynonymous P586L SNP modified the hPepT1 activity by decreasing in tenfold the transport capacity (Vmax) of the protein. The researches demonstrated that this decrease on transport capacity was due to a lower level of expression of hPepT1 and not because of intrinsic changes in transport function. Overall, these data suggest that Pro586 may have an important effect on hPepT1 translation, degradation, and/or membrane insertion.

Ishikawa et al. (2004) have published the identification of other potentially relevant nonsynonymous SNPs found in the Japanese population; however, no functional analysis has been conducted so far. The known nonsynonymous SNPs for hPepT1 can be observed in Table 7.6.

Table 7.6. Nonsynonymous polymorphisms in the PepTl gene (reference NM.005073)

NCBI SNP ID

dbSNP allele

Effect on protein

Functional relevancy

Not registered

C/T

P586L

Reduced transport capacity

(Zhang etal., 2004)

rs8187830

C/T

P537S

Unknown

rs2274827

C/Ta

R459C

None (Zhang et al., 2004)

rs8187838

C/A

T451N

None (Zhang et al., 2004)

rs2274828

G/Aa

V450I

None (Zhang et al., 2004)

rs4646227

G/Ca

G419A

None (Zhang et al., 2004)

Not registered

G/C

V416L

None (Zhang et al., 2004)

rs1782674

G/A

D383N

Unknown

aSNPs found in Japanese population (Ishikawa et al., 2004)

aSNPs found in Japanese population (Ishikawa et al., 2004)

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