Optimization of human nasal epithelium primary culture conditions for optimal proton oligopeptide and organic cation transporters expression in vitro.

AIM: To investigate the effect of key tissue culture conditions on cell growth, gene expression and functional uptake of peptide and organic cation transporter substrates in the human nasal epithelium (HNE). METHODS: HNE were cultured on different growth surfaces (polystyrene plastic, collagen film, and hydrated collagen gel) and were maintained with three popular nasal tissue culture media supplements [DMEM/F12 supplemented with Ultroser(®) G (2%), FBS (10%) and NuSerum(®) (10%)], respectively. The expression of gene transcripts for organic cation and peptide transporters were screened using qPCR and substrate uptake studies. RESULTS: Cell growth surface (polystyrene plastic surface, dried collagen film and hydrated collagen gel) did not significantly alter gene expression levels. However, Ultroser(®) G and FBS caused significant increase in PEPT1, PEPT2, PHT1, OCT3, and OCTN1 levels (~/=2-5-fold for FBS and 2-8-fold for Ultroser(®) G). In terms of the degree to which the supplements affected gene expression, the following observations were made: effect on OCTN1>PEPT2>OCT3>PHT1>PEPT1. Functional uptake of organic cation (4-Di-1-ASP) and peptide [ß-Ala-Lys (AMCA)] transporter substrates was significantly lower in cells cultured with NuSerum(®) compared to Ultroser(®) G and FBS cultured cells (p>0.05). CONCLUSIONS: Tissue culture media had a major effect on SLC gene expression levels of the human nasal epithelium in primary culture. Ultroser(®) G was identified as the most efficient culture supplement in maintaining SLC transporter expression under most culture conditions, whereas FBS appears to be an economical choice. We do not recommend the use of NuSerum(®) as a supplement for growing HNE for transport studies involving SLC transporters.

Proton-coupled oligopeptide transporter (POT) family expression in human nasal epithelium and their drug transport potential.

The molecular and functional expression of peptide transporters (PEPT1 and PEPT2, PHT1, PHT2) in human nasal epithelium was investigated. Quantitative/reverse transcriptase polymerase chain reaction (qPCR/RT-PCR), Western blotting and indirect immuno-histochemistry were used to investigate the functional gene and protein expression for the transporters. Uptake and transport studies were performed using metabolically stable peptides [ß-alanyl-L-lysyl-Nε-7-amino-4-methyl-coumarin-3-acetic acid (ß-Ala-Lys-AMCA) and ß-alanyl-L-histidine (carnosine)]. The effects of concentration, temperature, polarity, competing peptides, and inhibitors on peptide uptake and transport were investigated. PCR products corresponding to PEPT1 (150 bp), PEPT2 (127 bp), PHT1 (110 bp) and PHT2 (198 bp) were detected. Immunohistochemistry and Western blotting confirmed the functional expression of PEPT1 and PEPT2 genes. The uptake of ß-Ala-Lys-AMCA was concentration-dependent and saturable (Vmax =4.1 ( 0.07 µmol/min/mg protein, Km = 0.6 ( 0.07 µM). The optimal pH for intracellular accumulation of ß-Ala-Lys-AMCA was 6.5. Whereas dipeptides and carbonyl cyanide m-chlorophenylhydrazone (CCCP) significantly inhibited peptide uptake and transport, L-Phe had no effect on peptide transport. The permeation of ß-alanyl-L-histidine was concentration-, direction-, and temperature-dependent. The uptake, permeation, qPCR/RT-PCR and protein expression data showed that the human nasal epithelium functionally expresses proton-coupled oligopeptide transporters.

Differential expression of organic cation transporters in normal and polyps human nasal epithelium: implications for in vitro drug delivery studies.

The aim of this study was to compare the expression of organic cation transporters (OCTs) in normal and polyps nasal epithelium. Primary cell cultures of human nasal epithelium (polyps and normal tissues) were compared by investigating the uptake of a fluorescent organic cation, [4-dimethylaminostyryl-N-methylpyridinium (4-Di-1-ASP)]. The effect of concentration, temperature, pH and competing inhibitors were investigated. Quantitative polymerase chain reaction (qPCR) was used to compare the OCTs gene expression levels in the cells. The K(m) (µM) and V(max) (µM/mg protein/15 min) for 4-Di-1-ASP uptake were higher in normal (K(m)=3031 ± 559.6, V(max)=70.8 ± 8.8) cells compared to polyps (K(m)=952.4 ± 207.8, V(max)=30.9 ± 2.1). qPCR results showed that OCT1-3 and organic cation/carnitine transporter 1-2 gene transcripts (OCTN1-2) were expressed in both normal and polyps cells at comparable levels, with OCT-3 having the highest expression level in both cultures. Kruskal-Wallis ANOVA showed that pH and specific inhibitors had similar effects on both normal and polyps cells (p>0.5). Similarly, OCTs and OCTNs gene expression levels were similar. This study showed that polyps biopsies can be used for isolating cells to study organic cation transporters in human nasal epithelium as no major functional or molecular differences relative to normal cells could be found.

Paradoxical cyclosporine A requirements in pediatric renal transplants receiving high-dose steroids.

The potent immunosuppressant cyclosporine A (CyA) is a mainstay of treatment in the renal transplant population. During episodes of acute allograft rejection, therapy also includes the pulse administration of high-dose steroids such as prednisone or methylprednisolone. Both steroids and CyA are metabolized by the CYP3A4 isoenzyme of the cytochrome P450 catalytic system. On a theoretical basis, high steroid concentrations during a rejection episode could competitively inhibit CyA metabolism, increasing its systemic concentration and decreasing its dose requirements. A database was compiled consisting of pediatric patients who had undergone an acute renal rejection event during the years 1993 to 2003. The severity of rejection events, as well as the CyA and prednisone dosing regimens used during rejection, were assessed using a comprehensive chart analysis. The presence or absence of additional medications that could potentially interact with CyA was also examined. Although some patients responded in the predicted manner, the authors also found that a subgroup of pediatric patients placed on highdose pulse steroid therapy for acute graft rejection required increased amounts of CyA to maintain therapeutic concentrations. The authors recommend monitoring of patients on high-dose steroids for paradoxical CyA requirements intermittently during high-dose steroid treatment to individualize CyA therapy appropriately during renal allograft rejection and thereby maximize efficacy while minimizing potential toxic side effects of CyA such as under-immunosuppression and organ rejection.

Noradrenergic and serotonergic neuroendocrine responses in prepubertal, peripubertal, and postpubertal rats pretreated with desipramine and sertraline.

OBJECTIVE: To explore whether developmental status of neurotransmitter systems may affect response to antidepressant treatment. This study investigated whether younger animals, compared with mature animals, showed the same neuroendocrine response to challenge drug probes when pretreated with a serotonergic or noradrenergic antidepressant. METHOD: Prepubertal, pubertal, and adult rats were pretreated with low- or high-dose sertraline or desipramine for 14 days. Animals were then challenged with a noradrenergic probe (clonidine for desipramine-treated animals) or a serotonergic probe (fenfluramine for sertraline-treated animals). The neurohormonal response of growth hormone to the clonidine challenge and prolactin to the fenfluramine challenge was then measured. RESULTS: In animals challenged with fenfluramine, the postpubertal control group showed a significantly higher prolactin response to fenfluramine than postpubertal animals pretreated with low- or high-dose sertraline. No differences were found in the pubertal or prepubertal group. In animals challenged with clonidine, there was a significant age by treatment interaction effect for the prepubertal group pretreated with high doses of desipramine (less growth hormone secretion) but not for the peri- or postpubertal groups. CONCLUSIONS: These data indicate neurodevelopmental factors may play a role in the functional physiology of neurotransmitter systems, which in turn may affect response to psychotropics.