A novel role for PHT1 in the disposition of l-histidine in brain: In vitro slice and in vivo pharmacokinetic studies in wildtype and Pht1 null mice

Abstract

PHT1 (SLC15A4) is responsible for translocating l-histidine (l-His), di/tripeptides and peptide-like drugs across biological membranes. Previous studies have indicated that PHT1 is located in brain parenchyma, however, its role and significance in brain along with effect on the biodistribution of substrates is unknown. In this study, adult gender-matched Pht1-competent (wildtype) and Pht1-deficient (null) mice were used to investigate the effect of PHT1 on l-His brain disposition via in vitro slice and in vivo pharmacokinetic approaches. We also evaluated the serum clinical chemistry and expression levels of select transporters and enzymes in the two genotypes. No significant differences were observed between genotypes in serum chemistry, body weight, viability and fertility. PCR analyses indicated that Pept2 had a compensatory up-regulation in Pht1 null mice (about 2-fold) as compared to wildtype animals, which was consistent in different brain regions and confirmed by immunoblots. The uptake of l-His was reduced in brain slices by 50% during PHT1 ablation. The l-amino acid transporters accounted for 30% of the uptake, and passive (other) pathways for 20% of the uptake. During the in vivo pharmacokinetic studies, plasma concentration-time profiles of l-His were comparable between the two genotypes after intravenous administration. Still, biodistribution studies revealed that, when sampled 5 min after dosing, l-His values were 28–48% lower in Pht1 null mice, as compared to wildtype animals, in brain parenchyma but not cerebrospinal fluid. These findings suggest that PHT1 may play an important role in histidine transport in brain, and resultant effects on histidine/histamine homeostasis and neuropeptide regulation.

Introduction

PHT1 (SLC15A4), a member of the proton-coupled oligopeptide transporter (POT) superfamily, is responsible for translocating various di/tripeptides and peptide-like drugs across biological membranes, as well as the amino acid l-histidine [1]. Unlike two other peptide transporters, PEPT1 (SLC15A1) and PEPT2 (SLC15A2), which have been well studied, there is little known about PHT1 expression, localization, function and pharmacological relevance. PHT1 is abundantly found in the brain and eye [2], [3]. PHT1 has an age-dependent increase in brain expression [4], similar to the changes found in the histaminergic system [5], [6], [7], [8], [9]. In contrast, PEPT2 shows an age-dependent decrease in brain expression and PHT1 function dominated in the uptake of a dipeptide in brain slices from adult rodents, suggesting a significant role in regulating both endogenous and exogenous l-histidine (l-His) and peptides/mimetics in brain [4]. PHT1 transcripts are also detected in rat thyroid gland [10] and skeletal muscle [11]. A recent study showed that PHT1 and PHT2 are involved in the immune response [12]. These authors reported that PHTs were preferentially expressed in dendritic cells, located at endosomes and lysosomes, mediating the release of bacterially derived components into the cytosol.

l-Histidine (l-His) is one of the proteinogenic amino acids, which is obtained from dietary metabolism and protein turnover [13]. l-His possesses several crucial biological functions, including formation of the myelin sheath, detoxification of heavy metals, and the manufacturing of white and red blood cells. It is also a precursor of histamine, carnosine, ergothioneine, and vitamin C [14]. Studies on the transport of l-His in brain would extend our understanding of neuropeptide regulation, histamine homeostasis, and potential targets for drug delivery to neuronal and non-neuronal cells. In the central nervous system (CNS), major transporters for l-His include the Na⁺-coupled neutral amino acid transporters (e.g., SNATs, which belong to the SLC38 family) [15], [16], Na⁺-independent amino acid transporters (e.g., LATs and CATs, which belong to the SLC7 family) [17], [18], some members of the Na⁺- and Cl⁻-dependent neurotransmitter transporter family (e.g., B⁰AT2 and NTT4, which belong to the SLC6 family) [19], [20], [21], as well as the peptide/histidine transporters (e.g., PHT1 and PHT2, which belong to the SLC15 family) [4], [22].

Histamine, one of the most important metabolites of l-His, is formed in the brain during catalysis by histidine decarboxylase (HDC). While much of the research effort has focused on the enzymatic production of histamine via HDC, little attention has been paid to the mechanism by which l-His gains entry into neurons. Due to its polarity and lack of a transport system, histamine cannot pass through the barrier systems of the brain [23]. Moreover, the availability of its precursor, l-His, is positively correlated with brain histamine production and, consequently the regulatory functions of histamine [24], [25]. Significant changes in brain histamine levels have been observed in several neurological diseases, such as multiple sclerosis, Alzheimer’s disease, Down’s syndrome and Wernicke’s encephalopathy [26]. Dysfunction of HDC and the l-His transporters in brain may be related to these neurological disorders.

In the present study, we hypothesized that PHT1 ablation would substantially reduce the uptake of l-His in the brain of adult mice. Initial phenotypic analyses were performed in Pht1-competent (wildtype) and Pht1-deficient (null) mice, along with gene expression levels of POTs and select transporter/enzyme proteins. Subsequent studies evaluated the functional activity of PHT1 by studying the in vitro uptake of l-His in regional brain slices, and the in vivo pharmacokinetics and biodistribution of l-His after intravenous administration.