Urate transport capacity of glucose transporter 9 and urate transporter 1 in cartilage chondrocytes.

Chronic gouty arthritis, caused by a persistent increase in, and the deposition of, soluble uric acid (sUA), can induce pathological chondrocyte destruction; however, the effects of urate transport and intracellular sUA on chondrocyte functionality and viability are yet to be fully determined. Thus, the aim of the present study was to investigate the presence and functionality of a urate transport system in chondrocytes. The expression profiles of two primary urate reabsorptive transporters, glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1), in human articular cartilage and chondrocyte cell lines were examined via western blotting, reverse transcription­quantitative PCR, immunohistochemistry and immunofluorescence. Then, chondrocytes were incubated with exogenous sUA at increasing concentrations. Negative control assays were conducted via the specific knockdown of GLUT9 and URAT1 with lentiviral short hairpin (sh)RNAs, and by pretreatment with benzbromarone, a known inhibitor of the two transporters. Intracellular UA concentrations were measured using colorimetric assays. The expression levels of GLUT9 and URAT1 were determined in cartilage tissues and chondrocyte cell lines. Incubation of chondrocytes with sUA led to a concentration­dependent increase in intracellular urate concentrations, which was inhibited by GLUT9 or URAT1 knockdown, or by benzbromarone pretreatment (27.13±2.70, 44.22±2.34 and 58.46±2.32% reduction, respectively). In particular, benzbromarone further decreased the already­reduced intracellular UA concentrations in HC­shGLUT9 and HC­shURAT1 cells by 46.79±2.46 and 39.79±2.22%, respectively. Cells overexpressing GLUT9 and URAT1 were used as the positive cell control, which showed increased intracellular UA concentrations that could be reversed by treatment with benzbromarone. In conclusion, chondrocytes may possess an active UA transport system. GLUT9 and URAT1 functioned synergistically to transport UA into the chondrocyte cytoplasm, which was inhibited by specific gene knockdowns and drug­induced inhibition. These results may be fundamental in the further investigation of the pathological changes to chondrocytes induced by sUA during gouty arthritis, and identified UA transport processes as potential targets for the early control of chronic gouty arthritis.

Immunoreactivity of urate transporters, GLUT9 and URAT1, is located in epithelial cells of the choroid plexus of human brains.

It has been suggested that urate plays a protective role in neurons, while hyperuricemia is correlated with atherosclerosis and cardiovascular disease. However, whether there is a system that directly transports urate into the brain remains to be clarified. In this study, the localization of glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1), which are known to be representative reabsorptive urate transporters, was immunohistochemically examined in autopsied human brains. Immunoreactivity of GLUT9 was observed on the apical side of the cytoplasm of epithelial cells in the choroid plexus and in the cilia of ependymal cells of the human brain. Immunoreactivity of URAT1 was observed on the basolateral side of the cytoplasm of epithelial cells in the choroid plexus. In addition, immunoreactivity of GLUT9 and URAT1 was not observed in microvessels of the human brains. The choroid plexus and renal proximal tubule were similar in having a polarized distribution of these two transporters with the two transporters on opposite membranes, but the two transporters' distribution differs between the choroid plexus and the kidney in terms of which membrane (apical/basal) expresses which transporter. These findings support the hypothesis of the direct transport of intravascular urate into the central nervous system through the choroid plexus.

Quantitative Analysis of Human Red Blood Cell Proteome.

Red blood cells (RBCs) are the most abundant cell type in the human body. RBCs and, in particular, their plasma membrane composition have been extensively studied for many years. During the past decade proteomics studies have extended our knowledge on RBC composition; however, these studies did not provide quantitative insights. Here we report a large-scale proteomics investigation of RBCs and their "white ghost" membrane fraction. Samples were processed using the multienzyme digestion filter-aided sample preparation (MED-FASP) and analyzed using Q-Exactive mass spectrometer. Protein abundances were computed using the total protein approach (TPA). The validation of the data with stable isotope-labeled peptide-based protein quantification followed. Our in-depth analysis resulted in the identification of 2650 proteins, of which 1890 occurred at more than 100 copies per cell. We quantified 41 membrane transporter proteins spanning an abundance range of five orders of magnitude. Some of these, including the drug transporter ABCA7 and choline transporters SLC44A1 and SLC44A2, have not previously been identified in RBC membranes. Comparison of protein copy numbers assessed by proteomics showed a good correlation with literature data; however, abundances of several proteins were not consistent with the classical references. Because we validated our findings by a targeted analysis using labeled standards, our data suggest that some older reference data from a variety of biochemical approaches are inaccurate. Our study provides the first "in-depth" quantitative analysis of the RBC proteome and will promote future studies of erythrocyte structure, functions, and disease.

Organic cation transporter 3 (OCT3) is localized to intracellular and surface membranes in select glial and neuronal cells within the basolateral amygdaloid complex of both rats and mice.

Organic cation transporter 3 (OCT3) is a high-capacity, low-affinity transporter that mediates corticosterone-sensitive uptake of monoamines including norepinephrine, epinephrine, dopamine, histamine and serotonin. OCT3 is expressed widely throughout the amygdaloid complex and other brain regions where monoamines are key regulators of emotional behaviors affected by stress. However, assessing the contribution of OCT3 to the regulation of monoaminergic neurotransmission and monoamine-dependent regulation of behavior requires fundamental information about the subcellular distribution of OCT3 expression. We used immunofluorescence and immuno-electron microscopy to examine the cellular and subcellular distribution of the transporter in the basolateral amygdaloid complex of the rat and mouse brain. OCT3-immunoreactivity was observed in both glial and neuronal perikarya in both rat and mouse amygdala. Electron microscopic immunolabeling revealed plasma membrane-associated OCT3 immunoreactivity on axonal, dendritic, and astrocytic processes adjacent to a variety of synapses, as well as on neuronal somata. In addition to plasma membrane sites, OCT3 immunolabeling was also observed associated with neuronal and glial endomembranes, including Golgi, mitochondrial and nuclear membranes. Particularly prominent labeling of the outer nuclear membrane was observed in neuronal, astrocytic, microglial and endothelial perikarya. The localization of OCT3 to neuronal and glial plasma membranes adjacent to synaptic sites is consistent with an important role for this transporter in regulating the amplitude, duration, and physical spread of released monoamines, while its localization to mitochondrial and outer nuclear membranes suggests previously undescribed roles for the transporter in the intracellular disposition of monoamines.

Oct2 and Bob1 are sensitive and specific markers in lineage determination of B cell lymphomas with no expression of conventional B cell markers.

AIMS: Rare cases of B cell lymphomas do not express conventional B cell markers (CD20, CD79a and PAX5), and these types of lymphomas include anaplastic lymphoma kinase (ALK)-positive large B cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma and the solid variant of primary effusion lymphoma, extracavitary human herpesvirus 8 (HHV8)-positive large B cell lymphoma. Establishing accurate diagnoses of these B cell lymphomas can be challenging, and often requires a large panel of immunohistochemical stains, molecular assays and cytogenetic studies. B cell-specific transcription factors, Oct2 and Bob1, have been shown to be expressed consistently in most, if not all, B cell lymphomas, and therefore we investigated the utility of Oct2 and Bob1 immunohistochemistry in lineage determination of the aforementioned B cell lymphomas. METHODS AND RESULTS: We selected 34 cases of previously diagnosed B cell lymphomas with no or weak expression of CD20, CD79a and PAX5. Oct2 and Bob1 were positive in 74% (25 of 34) and 85% (29 of 34) of the cases, respectively. When we combined the results of these two immunostains, 94% (32 of 34) cases expressed at least one of these two markers. We also included 51 control cases of non-B cell neoplasms, and none of them expressed either Oct2 or Bob1. CONCLUSIONS: Oct2 and Bob1 are very reliable in determining B cell lineage in the absence of expression of other pan-B cell markers, and it should provide great diagnostic benefit to include them both in a panel of immunohistochemistry to assess undifferentiated malignant neoplasms.

MATEPRED-A-SVM-Based Prediction Method for Multidrug And Toxin Extrusion (MATE) Proteins.

The growth and spread of drug resistance in bacteria have been well established in both mankind and beasts and thus is a serious public health concern. Due to the increasing problem of drug resistance, control of infectious diseases like diarrhea, pneumonia etc. is becoming more difficult. Hence, it is crucial to understand the underlying mechanism of drug resistance mechanism and devising novel solution to address this problem. Multidrug And Toxin Extrusion (MATE) proteins, first characterized as bacterial drug transporters, are present in almost all species. It plays a very important function in the secretion of cationic drugs across the cell membrane. In this work, we propose SVM based method for prediction of MATE proteins. The data set employed for training consists of 189 non-redundant protein sequences, that are further classified as positive (63 sequences) set comprising of sequences from MATE family, and negative (126 sequences) set having protein sequences from other transporters families proteins and random protein sequences taken from NCBI while in the test set, there are 120 protein sequences in all (8 in positive and 112 in negative set). The model was derived using Position Specific Scoring Matrix (PSSM) composition and achieved an overall accuracy 92.06%. The five-fold cross validation was used to optimize SVM parameter and select the best model. The prediction algorithm presented here is implemented as a freely available web server MATEPred, which will assist in rapid identification of MATE proteins.

The pro-inflammatory cytokine tumor necrosis factor α stimulates expression of the carnitine transporter OCTN2 (novel organic cation transporter 2) and carnitine uptake via nuclear factor-κB in Madin-Darby bovine kidney cells.

Carnitine uptake into tissues is mediated mainly by the novel organic cation transporter 2 (OCTN2), whose expression is upregulated in the liver of early-lactating dairy cows. It has been shown recently that pro-inflammatory cytokines, including tumor necrosis factor α (TNFα), stimulate OCTN2 expression and carnitine uptake in intestinal cells and inflamed intestinal mucosa. Given that many early-lactating dairy cows show typical signs of hepatic and systemic inflammation, such as elevated concentrations of circulating TNFα and activation of the key regulator of inflammation, nuclear factor κB (NF-κB), in tissues, it is possible that upregulation of OCTN2 and increase of carnitine uptake by TNFα is mediated by NF-κB, a mechanism that might contribute to the upregulation of OCNT2 in the liver of early-lactating dairy cows. Thus, in the present study, we tested the hypothesis that TNFα stimulates OCTN2 gene expression and carnitine uptake via NF-κB in the bovine Madin-Darby bovine kidney (MDBK) cell line. Treatment with TNFα caused activation of NF-κB, increased the mRNA and protein concentration of OCTN2, and stimulated the uptake of carnitine in MDBK cells. In contrast, combined treatment of MDBK cells with TNFα and the NF-κB inhibitor BAY 11-7085 completely blocked the effect of TNFα on OCTN2 mRNA and protein concentration and uptake of carnitine. These findings suggest that the bovine OCTN2 gene and carnitine uptake are regulated by NF-κB. Future studies are required to show the in vivo relevance of this regulatory mechanism in cattle.

Precise comparison of protein localization among OCT, OAT, and MATE in human kidney.

Organic anion transporters (OATs) and organic cation transporters (OCT) play pivotal roles in the uptake of drugs into epithelial cells at the basolateral membranes, and multidrug and toxin extrusion (MATE) mediates drug secretion into urine at the brush-border membranes. In this study, the expression and distribution of apical MATE1 and MATE2-K, and basolateral OAT1, OAT3, and OCT2 were compared using serial sections of human kidney cortex. First, mRNA expression in the proximal tubules was evaluated using laser microdissection. Levels of OAT, OCT2, and MATE mRNA in the proximal tubules were greatly higher compared with glomerulus. The results quantitatively indicated that these transporters were localized to proximal tubules in the renal cortex. Second, MATE1 and MATE2-K protein were detected in proximal epithelial cells in which OCT2 protein was expressed at the basolateral membranes. In addition, MATE1 was expressed at the brush-border membranes of tubular epithelial cells in which OAT1 and OAT3 were expressed. The results confirmed that OAT1, OAT3, OCT2, MATE1, and MATE2-K were coexpressed in tubular epithelial cells. The cooperation among OAT, OCT, and MATE in renal drug secretion was consistent with their distribution.

Effect of lapatinib on hepatic parenchymal enhancement on gadoxetate disodium (EOB)-enhanced MRI scans of the rat liver.

PURPOSE: The purpose of this study was to investigate the effect of lapatinib treatment on hepatic parenchymal enhancement on Gd-EOB-MRI scans in rat. MATERIALS AND METHODS: Institutional animal review board approval was received prior to the commencement of all studies. Five rats received a single oral dose of 100 mg/kg/day lapatinib for 7 consecutive days. The controls (n = 5) were given 0.5 % (w/v) aqueous hydroxypropyl methyl cellulose containing 0.1 % (v/v) Tween 80 for 7 days. After the acquisition of gadoxetate disodium-enhanced MR images using 0.025 mmol gadolinium/kg, their livers were subjected to pathologic study to determine the expression level of organic anion-transporting polypeptide 1 (oatp1) and multi-drug resistance-associated protein 2 (mrp2). RESULTS: Relative enhancement of the liver was similar in both groups. At the hepatobiliary phase, which in rats occurs 3 min after the injection of Gd-EOB, it was 0.90 ± 0.06 in lapatinib-treated rats and 0.84 ± 0.08 in the controls (p = 0.30). There was also no difference in the expression level of oatp1 and mrp2. CONCLUSION: In rats, the administration of lapatinib for 7 days had no effect on hepatic parenchymal enhancement on Gd-EOB-MRI scans.

Differential expression of organic cation transporter OCT-3 in oral premalignant and malignant lesions: potential implications in the antineoplastic effects of metformin.

BACKGROUND: Recent evidence indicates that metformin, a biguanide used as first-line treatment for type 2 diabetes, prevents the conversion of carcinogen-induced oral dysplasias into head and neck squamous cell carcinomas (HNSCC), most likely by inhibiting mammalian target of rapamycin complex 1 (mTORC1) oncogenic signaling. Whether metformin acts directly at the primary tumor site or indirectly by modulating hormonal secretion from extratumoral organs remains unknown. As organic cation transporters (OCT) belonging to the solute carrier 22A gene family, including OCT-1, OCT-2, and OCT-3, mediate metformin uptake and activity, it is critical to define what role they play in the antineoplastic activity of metformin. METHODS: Immunohistochemical and immunoblotting techniques were used in normal, dysplastic and HNSCC tissues, and HNSCC cell lines, respectively, to determine OCTs expression levels. RESULTS: We report that only OCT-3 was highly expressed in a number of HNSCC cell lines, oral epithelial dysplasias, and well to moderately differentiated HNSCC. Indeed, inhibition of OCT-3 expression and activity in HNSCC cells prevented metformin-induced AMP-activated protein kinase activation and mTORC1 pathway inhibition. Moreover, in oral dysplasias, high OCT-3 expression localized to epithelial compartments where mTORC1 signaling was also upregulated suggestive of a potential local effect of metformin. CONCLUSIONS: The concept of using metformin as a chemopreventive agent to control head and neck carcinogenesis is promising. Further work is warranted to elucidate largely unexplored mechanisms of metformin uptake and pharmacologic action that may ultimately influence the selection of the most suitable patients who can benefit from metformin in head and neck cancer chemoprevention.

NGAL and NGALR overexpression in human hepatocellular carcinoma toward a molecular prognostic classification.

AIM: Neutrophil gelatinase-associated lipocalin (NGAL) and its cell surface receptor, NGALR, have been implicated in tumorigenesis and tumor progression of various human malignant neoplasms. In particularly, it has been demonstrated that NGAL is overexpressed in hepatocellular carcinoma (HCC) tissues and closely associated with the proliferation and invasion of HCC cells. The aim of this study was to investigate the clinical significance of NGAL and NGALR in HCC. METHODS: Expression of NGAL and NGALR was evaluated by immunohistochemistry in tumor tissues from 138 patients who underwent curative resection of HCC. The association of NGAL or NGALR expression with the clinicopathologic features was analyzed. Univariate and multivariate analyses were performed to evaluate the prognostic value of NGAL and/or NGALR expression for HCC patients. RESULTS: The expression levels of NGAL and NGALR were both up-regulated in HCC tissues, and to be associated with vascular invasion (both P=0.03), TNM stage (both P=0.004), and tumor recurrence (both P<0.001). A positive correlation between expression of the two markers was also observed (r=0.89; P<0.001). Additionally, survival analysis showed that high expression of NGAL or NGALR was significantly associated with poor prognosis for patients with HCC (both P=0.003). Patients with high expression of both NGAL and NGALR had a shorter overall survival (P<0.001) than those with low expression of both. Furthermore, multivariate analysis showed both NGAL and NGALR were independent predictors of overall survival. CONCLUSION: Our data demonstrate for the first time that the up-regulations of NGAL and NGALR expression in HCC were both significantly correlated with unfavorable clinicopathologic features and independent poor prognostic factor for overall survival in patients. These findings suggest that NGAL and NGALR expression might be served as novel prognostic factors and potential therapeutic targets in HCC.

Alterations in the non-neuronal acetylcholine synthesis and release machinery in esophageal epithelium.

AIMS: A non-neuronal cholinergic system has been described in epithelial cells including that of the urinary bladder (urothelium) and the upper gastrointestinal tract (esophagus). Epithelial dysfunction has been implicated in the pathophysiology of persistent pain conditions such as painful bladder syndrome as well as functional heartburn. For example, alterations in the ability to synthesize and release acetylcholine may contribute to changes in epithelial sensory and barrier function associated with a number of functional genitourinary and intestinal disorders. MAIN METHODS: We examined using immunoblot, acetylcholine (ACh)-synthesis and release components in cat esophageal mucosa and whether elements of these components are altered in a naturally occurring model of chronic idiopathic cystitis termed feline interstitial cystitis (FIC). KEY FINDINGS: We identified proteins involved in ACh synthesis and release (high affinity choline transporter, CHT1; ACh synthesizing enzyme choline acetyltransferase ChAT and carnitine acetyltransferase CarAT; vesicular ACh transporter VAChT and the organic cation transporter isoforms 1-3 or OCT-1-3) in cat esophageal mucosa. Significant alterations in CHT, ChAT, VAChT and OCT-1 were detected in the esophageal mucosa from FIC cats. Changes in the vesicular nucleotide transporter (VNUT) and the junctional protein pan-cadherin were also noted. SIGNIFICANCE: Taken together, these findings suggest that changes in the non-neuronal cholinergic system may contribute to alterations in cell-cell contacts and possibly communication with underlying cells that may contribute to changes in sensory function and visceral hyperalgesia in functional esophageal pain.

Does bladder outlet obstruction alter the non-neuronal cholinergic system of the human urothelium?

AIMS: Alterations of the bladder sensory system are considered to contribute to detrusor overactivity (DO) when patients suffer from bladder outlet obstruction (BOO). The urothelium is one part of this sensory system and it harbors a non-neuronal cholinergic system (NNCS). We aimed to investigate if BOO causes alterations in the NNCS. MAIN METHODS: Urothelial specimens were collected by endoscopy from six male controls and eight male patients suffering from BOO and DO. The samples were examined by immunofluorescence (IF) and real-time RT-PCR for high-affinity choline transporter-1 (CHT1), choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), organic cation transporters OCT1-3, muscarinic receptor (mAChR) subtypes M1-M5 and nicotinic receptor (nAChR) subunits α7, α9 and α10. KEY FINDINGS: ChAT, VAChT and OCT2 are not present in the male urothelium. Real-time RT-PCR and IF detected all other investigated targets. Rank order of expression was M2≫M3=M5>M4=M1 for mAChR subtypes and α7≫α10>α9 for nAChR subunits. Statistical analysis of RT-PCR results did not detect significant differences between patients and controls. Only IF detected differences between both groups: α9-Immunolabeling was increased in all BOO/DO patients. SIGNIFICANCE: BOO does not induce considerable alterations of the human urothelial NNCS on mRNA level. Expression of mAChRs, CHT1, OCT1 and OCT3 is not significantly affected by BOO. Thus, transport mechanisms for choline and acetylcholine (ACh) stay unaltered. BOO increases immunolabeling of α9-nAChR but whether this sole finding contributes to the onset of DO seems questionable. Comparing the present results with our previous work, the urothelial NNCS does not differ between men and women.

Gene expression analysis of membrane transport proteins in normal and lipopolysaccharide-inflamed rat dental pulp.

INTRODUCTION: Membrane transport proteins (transporters) play a crucial role in the transmembrane uptake and/or efflux of various compounds such as inorganic ions, endogenous bioactive substances such as prostaglandins (PGs), and drugs such as nonsteroidal anti-inflammatory drugs. This study aimed to analyze mRNA expression of selected transporters related to drug disposition and PG transport in normal and lipopolysaccharide (LPS)-inflamed rat incisor pulp. METHODS: Pulp tissues were subjected to reverse transcription-polymerase chain reaction (PCR) detection for transporter isoforms belonging to organic anion transporting polypeptide (Oatp), organic anion transporter (Oat), organic cation transporter (Oct), multidrug resistance-associated protein (Mrp), and multidrug resistance protein (Mdr) families. The levels of mRNA expression for PG transporters (Oatp1a5, Oatp1b2, Oatp2a1, Oatp2b1, and Oatp3a1) were compared in normal and LPS-inflamed pulps by using real-time PCR. RESULTS: The pulp tissue expressed mRNAs for various transporters belonging to the Oatp, Oat, Oct, Mrp, and Mdr families. LPS inflammation caused significant up-regulation of Oatp2a1 (P < .01) and significant down-regulation of Oatp1a5, Oatp2b1 (P < .01), and Oatp3a1 (P < .05). CONCLUSIONS: Rat incisor dental pulp expressed mRNAs for various transporter isoforms. The levels of mRNA expression for PG transporters were significantly up-regulated or down-regulated in LPS-inflamed dental pulp.

Elevated systemic elimination of cimetidine in rats with acute biliary obstruction: the role of renal organic cation transporter OCT2.

Renal tubular secretion of cationic drugs is dominated by two classes of organic cation transporters, OCT2/SLC22A2 and MATE1/SLC47A1, localized to the basolateral and brush-border membranes of the renal tubular epithelial cells, respectively. However, little is known about the expression and function of these transporters in acute cholestasis. Systemic clearance of cimetidine was significantly higher in rats with bile duct ligation (BDL) for 24 hours than in sham-operated rats, with no significant changes in the volume of distribution between the groups. In addition, net tubular secretory clearance of cimetidine was significantly higher in the BDL rats compared with the sham rats, with no significant changes in the glomerular filtration rate. Moreover, the renal tissue-to-plasma concentration ratio of cimetidine was elevated in BDL rats, although the renal tissue-to-urine clearance ratio of cimetidine was not different between the two groups. The expression level of basolateral organic cation transporter rOCT2 protein in the kidney cortex was markedly higher in BDL rats than that in the sham rats, but that of H+/organic cation antiporter rMATE1 protein in the brush-border membranes was not significantly different between the two groups. These results demonstrate that the renal tubular secretion of cimetidine was increased by acute cholestasis, and this increase was attributable to elevated expression levels of rOCT2 but not of rMATE1 in the rat.

Transport of carnitine and acetylcarnitine by carnitine/organic cation transporter (OCTN) 2 and OCTN3 into epididymal spermatozoa.

Carnitine and acetylcarnitine are important for the acquisition of motility and maturation of spermatozoa in the epididymis. In this study, we examined the involvement of carnitine/organic cation transporter (OCTN) in carnitine and acetylcarnitine transport in epididymal spermatozoa of mice. Uptake of both compounds by epididymal spermatozoa was time-dependent and partially Na(+)-dependent. Kinetic analyses revealed the presence of a high-affinity transport system in the spermatozoa, with K(m) values of 23.6 and 6.57 muM for carnitine and acetylcarnitine respectively in the presence of Na(+). Expression of OCTN2 and OCTN3 in epididymal spermatozoa was confirmed by immunofluorescence analysis. The involvement of these two transporters in carnitine and acetylcarnitine transport was supported by a selective inhibition study. We conclude that both Na(+)-dependent and -independent carnitine transporters, OCTN2 and OCTN3, mediate the supply of carnitine and acetylcarnitine to epididymal spermatozoa in mice.

The effect of corticosteroids on the disposal of long-acting beta2-agonists by airway smooth muscle cells.

BACKGROUND: Organic cation transporters (OCTs) have an important role in tissue distribution and elimination of cationic drugs. Carrier-mediated disposal of cationic bronchodilators in the airway tissue, however, is incompletely understood. OBJECTIVES: We sought to assess the uptake of long-acting beta(2)-agonist bronchodilators by bronchial and vascular smooth muscle cells. METHODS: Human airway cells and tissues obtained from organ donors were evaluated for cationic drug transporter expression by means of quantitative RT-PCR and immunofluorescence. For in vitro functional studies, tritiated formoterol and tritiated salmeterol uptake by bronchial and vascular smooth muscle cells was measured. RESULTS: Quantitative RT-PCR analysis revealed high mRNA levels for the corticosteroid-sensitive OCT3 in bronchial and vascular smooth muscle cells. Immunofluorescence staining of airway sections confirmed OCT3 expression in these cells. In bronchial smooth muscle cells, uptake of the cationic formoterol was inhibited with OCT inhibitors. Corticosteroids also inhibited formoterol uptake through a rapid (within 15 minutes) nongenomic action, with the following rank order for inhibitory potency: corticosterone > budesonide > fluticasone. The corticosteroid-induced inhibition was significantly higher in vascular than bronchial smooth muscle cells. In comparison with formoterol, uptake of the noncharged lipophilic salmeterol was approximately 10-fold higher and insensitive to all OCT inhibitors and corticosteroids. CONCLUSIONS: Our findings suggest that corticosteroids, through OCT3 inhibition, rapidly interfere with the disposal of cationic drugs by smooth muscle cells in the airway. CLINICAL IMPLICATIONS: This novel immediate interaction between corticosteroids and cationic beta(2)-agonist bronchodilators supports the use of such combinations in the pharmacotherapy of asthma.

Influence of estrogen and xenoestrogens on basolateral uptake of tetraethylammonium by opossum kidney cells in culture.

The sex steroid hormone estrogen down-regulates renal organic cation (OC) transport in animals, and it may contribute to sex-related differences in xenobiotic accumulation and excretion. Also, the presence of various endocrine-disrupting chemicals, i.e., environmental chemicals that possess estrogenic activity (e.g., xenoestrogens) may down-regulate various transporters involved in renal accumulation and excretion of xenobiotics. The present study characterizes the mechanism by which long-term (6-day) incubation with physiological concentrations of 17beta-estradiol (E(2)) or the xenoestrogens diethylstilbestrol (DES) and bisphenol A (BPA) regulates the basolateral membrane transport of the OC tetraethylammonium (TEA) in opossum kidney (OK) cell renal cultures. Both 17beta-E(2) and the xenoestrogen DES produced a dose- and time-dependent inhibition of basolateral TEA uptake in OK cell cultures, whereas the weakly estrogenic BPA had no effect on TEA uptake. Treatment for 6 days with either 1 nM 17beta-E(2) or DES reduced TEA uptake by approximately 30 and 40%, respectively. These effects were blocked completely by the estrogen receptor antagonist ICI 182780 (Faslodex, fulvestrant), suggesting that these estrogens regulate OC transport through the estrogen receptor, which was detected (estrogen receptor alpha) in OK cell cultures by reverse transcription-polymerase chain reaction. The J(max) value for TEA uptake in 17beta-E(2)- and DES-treated OK cell cultures was approximately 40 to 50% lower than for ethanol-treated cultures, whereas K(t) was unaffected. This reduction in transport capacity was correlated with a reduction in OC transporter OCT1 protein expression following treatment with both agents.

Comparative gene expression profiles of intestinal transporters in mice, rats and humans.

We have studied gene expression profiles of intestinal transporters in model animals and humans. Total RNA was isolated from duodenum and the mRNA expression was measured using Affymetrix GeneChip oligonucleotide arrays. Detected genes from the intestine of mice, rats, and humans were about 60% of 22,690 sequences, 40% of 8739, and 47% of 12,559, respectively. A total of 86 genes involving transporters expressed in mice, 50 genes in rats, and 61 genes in humans were detected. Mice exhibited abundant mRNA expressions for peptide transporter HPT1, amino acid transporters CSNU3, CT1 and ASC1, nucleoside transporter CNT2, organic cation transporter SFXN1, organic anion transporter NBC3, glucose transporter SGLT1, and fatty acid transporters FABP1 and FABP2. Rats showed high expression profiles of peptide transporter PEPT1, amino acid transporters CSNU1 and 4F2HC, nucleoside transporter CNT2, organic cation transporter OCT5, organic anion transporter SDCT1, glucose transporter GLUT2 and GLUT5, and folate carrier FOLT. In humans, the highly expressed genes were peptide transporter HPT1, amino acid transporters LAT3, 4F2HC and PROT, nucleoside transporter CNT2, organic cation transporter OCTN2, organic anion transporters NADC1, NBC1 and SBC2, glucose transporters SGLT1 and GLUT5, multidrug resistance-associated protein RHO12, fatty acid transporters FABP1 and FABP2, and phosphate carrier PHC. Overall these data reveal diverse transcriptomic profiles for intestinal transporters among these species. Therefore, this transcriptional data may lead to more effective use of the laboratory animals as a model for oral drug development.

Low-affinity uptake of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (4-Di-1-ASP) in BeWo cells.

Understanding the mechanisms of transport processes in the placenta can improve the safety and efficacy of drug delivery during pregnancy. Functional studies of organic cation transporters (OCTs) are usually carried out using radioactivity, and a fluorescent marker would add flexibility to experimental methods. As a published substrate for OCT1 and OCT2, the fluorescent compound 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (4-Di-1-ASP) was chosen as a candidate for studying placental OCT function in BeWo cells. The expression of OCT1 and OCT2 was also investigated in BeWo cells, an established human choriocarcinoma trophoblastic cell line frequently used as an in vitro model of the rate-limiting barrier for maternal-fetal exchange of drugs and nutrients within the placenta. 4-Di-1-ASP was taken up into BeWo cells by a low-affinity, carrier-mediated process exhibiting a Km of 580+/-110 microM and Vmax of 97+/-9 nmol/mg protein/30 min, and asymmetric transport was observed, with greater permeability in the apical to basolateral (maternal-to-fetal) direction. However, RT-PCR revealed no expression of OCT1 or OCT2 in either BeWo cells or primary cultured human cytotrophoblast cells, and OCT substrates such as TEA and choline did not inhibit the uptake of 4-Di-1-ASP. Although the uptake of this fluorescent compound in BeWo cells is not mediated by an OCT, the colocalization experiments with fluorescence microscopy and inhibition studies confirmed significant mitochondrial uptake of 4-Di-1-ASP. Transport of 4-Di-1-ASP into the nuclear region of BeWo cells was also observed, which is likely mediated by a nucleoside transporter.