Kinetic Analysis of p-Aminohippurate Transport in the OK Kidney Epithelial Cell Line

Pharmaceutical Research -     

Transport of Levofloxacin in the OK Kidney Epithelial Cell Line: Interaction with p-Aminohippurate Transport

Purpose. To evaluate the mechanism of renal transport of quinolone antibacterial drugs, we examined the interaction of levofloxacin with p–aminohippurate (PAH) transport systems and the transport of levofloxacin in renal epithelial cells. Methods. Transport of [¹⁴C]PAH or [¹⁴C]levofloxacin was measured using OK cell monolayers grown on microporous membrane filters. Results. Transcellular transport from the basolateral to the apical side and cellular accumulation of [¹⁴C]PAH were inhibited by levofloxacin. Both the initial uptake of [¹⁴C]PAH from the basolateral side and the efflux to the apical side were inhibited by levofloxacin. The basolateral–to–apical transcellular transport of [¹⁴C]levofloxacin was greater than that in the opposite direction. [¹⁴C]Levofloxacin efflux to the apical side was greater than that to the basolateral side. Unlabeled levofloxacin and grepafloxacin inhibited the transcellular transport of [¹⁴C]levofloxacin, accompanied by an increase of cellular accumulation. However, neither PAH nor an anion transport inhibitor 4–4–diisothiocyanostilbene–2,2–disulfonic acid (DIDS) affected the basolateral–to–apical transport of [¹⁴C]levofloxacin nor its uptake from the basolateral side. Conclusions. These results indicated that levofloxacin inhibits PAH transport across both the basolateral and apical membranes of OK cells, but are not transported via the systems for PAH transport. The existence of a specific transport system for quinolones was indicated in OK cells.     

Effect of Substituted Benzoylglycines (Hippurates) and Phenylacetylglycines on p-Aminohippurate Transport in Dog Renal Membrane Vesicles

The effect of substituted benzoylglycines (hippurates) and phenylacetylglytines on the transport of p-aminohippurate (PAH) was studied in basolateral (BLMV) and brush border membrane vesicles (BBMV) isolated from dog kidney cortex. The probenecid-sensitive part of 100 µM [³H]PAH uptake into BLMV and BBMV was measured in the presence and absence of 5 mM glycine conjugate. The benzoyl- and phenylacetylglycines studied were substituted in the 2-, 3-, or 4-position with an H, CH₃, OCH₃ or OH group. Benzoylglycines were stronger inhibitors of PAH transport than phenylacetylglycines and the inhibitory potency of the conjugates was in general lower against the transporter in BBMV than BLMV. The specificities of the transporters in both membranes appear to be very similar. The inhibitory potency of the benzoylglycines, expressed as the apparent inhibition constant (logK _i), did not show a linear relationship with their lipophilicity as determined by reversed phase HPLC. Deviation from linearity was caused mainly by the 3-OH and 4-OH analogs, which showed a greater inhibitory potency than expected from their lipophilicity. Phenylacetylglycines only showed a small variation in logK _i, values, indicating that insertion of a CH₂ group between the ring and the carbonyl practically abolishes the influence of the ring and its substituents. In conclusion, both hydrophobic and electronic properties are important determinants of affinity for the PAH transport system. An additional partially negative hydroxyl group in the ring, located preferably at the 3- or 4-position, increases the interaction with the transport system.     

Kinetic Analysis of Tetraethylammonium Transport in the Kidney Epithelial Cell Line,LLC-PK1

Purpose. The aims of this study were to establish a kinetic means of analyzing the membrane transport of organic cations in renal epithelial cells, and to simultaneously evaluate drug interactions in apical and basolateral membranes. Methods. Tetraethylammonium (TEA) transport was measured using LLC-PK₁, cell monolayers grown on microporous membrane filters. After incubating the cells with unlabeled TEA or other drugs, apical or basolateral medium was changed to that containing labeled TEA, and transcellular transport and cellular accumulation were measured. Clearance from apical medium to cells (CL₁₂), cells to apical medium (CL₂₁), cells to basolateral medium (CL₂₃) and basolateral medium to cells (CL₃₂) were calculated based on a three compartment model. Results. TEA was accumulated progressively in the monolayers from the basolateral side and was transported unidirectionally to the apical side. CL₃₂ was greater than CL₁₂ and CL₂₃ was greater than CL₂₁. Therefore, the rate limiting step of TEA transport from the basolateral to the apical medium was the cell-to-apical step. Co-incubation of TEA with procainamide decreased the transport parameters of TEA, CL₁₂, CL₂₁ and CL₃₂, whereas that with levofloxacin decreased only CL₁₂ and CL₂₁, not affecting the parameters in basolateral membranes. Conclusions. Using a simple model, we analyzed the transport of organic cation in kidney epithelial cell line, LLC-PK₁ This method can be useful for the analysis of cation transport and drug interactions in the apical and basolateral membranes of renal tubules.     

Functional Identification of a Novel Transport System for Endogenous and Synthetic Opioid Peptides in the Rabbit Conjunctival Epithelial Cell Line CJVE

Purpose  To investigate whether conjunctival epithelial cells express transport processes for opioid peptides. Methods  We monitored the uptake of [³H]deltorphin II and [³H]DADLE, two hydrolysis-resistant synthetic opioid peptides, in the rabbit conjunctival epithelial cell line CJVE and elucidated the characteristics of the uptake process. Results  CJVE cells express robust uptake activity for deltorphin II and DADLE. Both opioid peptides compete with each other for transport. Several endogenous and synthetic opioid peptides, but not non-peptide opioid antagonists, are recognized by the transport process. Though various peptides inhibit the uptake of deltorphin II and DADLE in a similar manner, the uptake of deltorphin II is partly Na⁺-dependent whereas that of DADLE mostly Na⁺-independent. The transport process shows high affinity for many endogenous/synthetic opioid peptides. Functional features reveal that this transport process may be distinct from the opioid peptide transport system described in the retinal pigment epithelial cell line ARPE-19 and also from the organic anion transporting polypeptides, which are known to transport opioid peptides. Conclusions  CJVE cells express a novel, hitherto unknown transport process for endogenous/synthetic opioid peptides. This new transport process may offer an effective delivery route for opioid peptide drugs to the posterior segment of the eye.     

Organic Cation Transport in Rabbit Alveolar Epithelial Cell Monolayers

Purpose. To characterize organic cation (OC) transport in primary cultured rabbit alveolar epithelial cell monolayers, using [^l4C]-guanidine as a model substrate. Methods. Type II alveolar epithelial cells from the rabbit lung were isolated by elastase digestion and cultured on permeable filters pre-coated with fibronectin and collagen. Uptake and transport studies of [¹⁴C]-guanidine were conducted in cell monolayers of 5 to 6 days in culture. Results. The cultured alveolar epithelial cell monolayers exhibited the characteristics of a tight barrier. [¹⁴C]-Guanidine uptake was temperature dependent, saturable, and inhibited by OC compounds such as amiloride, cimetidine, clonidine, procainamide, propranolol, tetraethyl-ammonium, and verapamil. Apical guanidine uptake (K_m = 129 ± 41 M, V_max = 718 ± 72 pmol/mg protein/5 min) was kinetically different from basolateral uptake (K_m = 580 ± 125 (M, V_max = 1,600 ± 160 pmol/mg protein/5 min). [¹⁴C]-Guanidine transport across the alveolar epithelial cell monolayer in the apical to basolateral direction revealed a permeability coefficient (P_app) of (7.3 ± 0.4) × 10^-7 cm/sec, about seven times higher than that for the paracellular marker [¹⁴C]-mannitol. Conclusions. Our findings are consistent with the existence of carrier-mediated OC transport in cultured rabbit alveolar epithelial cells.     

Transepithelial Transport of Diphenhydramine Across Monolayers of the Human Intestinal Epithelial Cell Line Caco-2

Purpose. The transepithelial transport characteristics of theantihistamine, diphenhydramine, were studied in human intestinal Caco-2 cellmonolayers to elucidate the mechanisms of its intestinal absorption. Methods. The transepithelial transport and the cellular accumulationof diphenhydramine were measured using Caco-2 cell monolayersgrown in Transwell chambers. Results. The transepithelial transport of diphenhydramine from theapical to basolateral side was saturable, and the flux and cellularaccumulation of diphenhydramine were dependent on the apicalextracellular pH (pH 7.4 > 6.5 > 5.5). Transport and accumulation ofdiphenhydramine from the apical side were inhibited by anotherantihistamine, chlorpheniramine, while typical substrates for the renal organiccation transport system such as tetraethylammonium, cimetidine andguanidine had no effect. The transepithelial transport and cellularaccumulation of diphenhydramine from the basolateral side were alsopH-dependent and inhibited by chlorpheniramine. In addition, intracellulardiphenhydramine preloaded was preferentially effluxed to the apicalside, suggesting the involvement of the secretory pathway indiphenhydramine transport. Furthermore, diphenhydramine uptake from boththe apical and basolateral sides was stimulated by preloadingmonolayers with chlorpheniramine (trans-stimulation effect). Conclusions. Transepithelial transport of diphenhydramine acrossCaco-2 cells is mediated by pH-dependent, specific transport systemsthat exist in both the apical and basolateral membranes.     

Competitive Inhibition of p-Aminohippurate Transport by Quinapril in Rabbit Renal Basolateral Membrane Vesicles

The mechanism of quinapril's interaction with the organic anion transporter was characterized by studying its effect on the transport of p-aminohippurate (PAH) in rabbit renal basolateral membrane vesicles (BLMV). Cis-inhibition studies demonstrated that quinapril was a specific and potent inhibitor of PAH. The K_i of quinapril was about 20 M, a value similar to that of probenecid and eight-times lower than the K_m value of 165 M for PAH. Even though quinapril resulted in trans-inhibition of PAH uptake during counterflow studies, kinetic studies revealed that quinapril was a competitive inhibitor of PAH transport. This latter finding suggests that quinapril and PAH share a common binding site on the transporter. Overall, the results indicate that quinapril is a high-affinity inhibitor of the organic anion transporter in renal BLMV, and that drug–drug interactions may occur with other organic anions at the basolateral membrane of proximal cells.     

The Madin Darby Canine Kidney (MDCK) Epithelial Cell Monolayer as a Model Cellular Transport Barrier

Two strains of Madin Darby canine kidney (MDCK) cells were grown on a polycarbonate membrane with 3-µm pores without any extracellular matrix treatment. The membrane, 2.45 cm in diameter, which is part of a commercially obtained presterilized culture insert, provides two chambers when placed in a regular six-well culture plate. This device was found to be convenient for investigating transport of a few selected fluid-phase markers across the MDCK cell monolayer. Both the strain from the American Type Culture Collection (ATCC) and the so-called highly resistant strain I, at a serial passage between 65 and 70, showed a seeding concentration-dependent lag phase followed by a growth phase with a 21-hr doubling time. When seeded at 5 × 10⁴ cells/cm², cell confluence was achieved in 5 days in a modified Eagle's minimum essential medium (MEM) containing 10% fetal bovine serum under a 5% CO₂ atmosphere. Similarly, transepithelial electrical resistance (TEER) also reached a plateau value in 5 days. Both light and electron microscopic examinations revealed well-defined junctional structures. Transport of the fluid-phase markers, sucrose, lucifer yellow CH (LY), inulin, and dextran across the MDCK cell monolayers was studied primarily at 37°C following the apical-to-basolateral as well as the basolateral-to-apical direction. Large variations in the steady-state transport rate were observed for a given marker between the cell layer preparations. Thus, the present study proposes an internal standard procedure for meaningful comparisons of the transport rate. When normalized to the rate of sucrose, the rate ratio was 1.00:0.80:0.67:0.15 for sucrose:LY:inulin:dextran. This ratio was virtually independent of temperature, cell strain, direction of the marker migration, and TEER value, suggesting a common transport mechanism. The observed rate ratio appears to reflect molecular size and charge. The transport observed in the present study would consist, in theory, of both paracellular shunt and transcellular vesicular transport. Quantitative assessment of each transport mechanism in the overall transport has been difficult. The initial uptake of [³H]dextran estimated for the slowest transport observed in the present study was still 300-fold faster than a literature value. This appears to indicate that the transport observed in the present study is largely through the paracellular shunt pathway.     

Neutrophil-Mediated Transport of Liposomes Across the Madin Darby Canine Kidney Epithelial Cell Monolayer

Targeted drug delivery to peripheral blood neutrophils (PMNs) should be of therapeutic potential in various disease states. In addition, substances taken up by PMNs in the circulation may be delivered to an extravascular site via the naturally occurring cell infiltration. The present study employs an in vitro chemotaxis model to test whether particulate drug carriers such as liposomes can be transported across a cellular barrier by migrating PMNs. The system contained 10⁷ human PMNs/ml, 0.3-µm liposomes at a total lipid concentration of 2.5 mM, and 10% autologous human serum in the apical side of a confluent Madin Darby canine kidney (MDCK) epithelial cell monolayer of 4.71 cm². The MDCK cells were grown on a polycarbonate membrane with 3-µm pores without any extracellular matrix, and 10^–7 M f-Met-Leu-Phe was added to the basolateral side as a trigger of chemotaxis. The aqueous phase of the reverse-phase evaporation vesicles (REVs) contained lucifer yellow CH (LY) and [¹⁴C]sucrose. The lipid bilayer of the REVs was spiked with [³H]dipalmitoylphosphatidylcholine (DPPC). Transmission electron micrographs showed that, in response to the formyl peptide, PMNs adhered to the apical surface of MDCK cells, emigrated across the MDCK cell layer, passed through the 3-µm pores in the polycarbonate membrane, and finally, appeared in the bottom well. Epifluorescence micrographs showed that most, if not all, of the migrated PMNs contained punctate fluorescence derived from LY. Transport data over a 3.5-hr period indicated that those markers that appeared in the basal side were indeed transported by phagocytosis of REVs by PMNs and that intact serum was an essential component in the process. The PMN-mediated transport of REVs may serve as a possible targeted drug delivery to an extravascular site in vivo in various inflammatory diseases.     

Decreased Transport of p-Aminohippurate in Renal Basolateral Membranes Isolated from Rats with Acute Renal Failure

Transport of D-glucose, p-aminohippurate (organic anion), and tetraethylammonium (organic cation) was studied in the renal basolateral membrane vesicles isolated from rats with acute renal failure (ARF). ARF was induced by a single injection of uranyl nitrate. Carrier-mediated transport of p-aminohippurate, estimated under anion–anion exchange condition, was significantly decreased in basolateral membrane vesicles isolated from ARF rats. In contrast, there were no significant differences in D-glucose and tetraethylammonium uptake between normal and ARF rats. When normal basolateral membrane vesicles were incubated in vitro with uranyl nitrate, no significant inhibition in p-aminohippurate uptake was observed. These results suggest that organic anion transport is decreased in renal basolateral membranes from ARF rats, and this transport dysfunction cannot be explained by the direct interaction of uranyl nitrate with the organic anion carrier.     

Dipeptide Uptake and Transport Characteristics in Rabbit Tracheal Epithelial Cell Layers Cultured at an Air Interface

Purpose. To determine the functional presence of a H⁺/peptide cotransport process in rabbit tracheal epithelial cell layers cultured at an air-interface and its contribution to transepithelial dipeptide transport. Methods. Rabbit tracheocytes were isolated, plated on Transwells, and cultured at an air-interface. After 5 or 6 days in culture, uptake and transepithelial transport of carnosine were examined. Results. Carnosine uptake by tracheocytes was pH-dependent and was saturable with a Michaelis-Menten constant of 170 M. Moreover, carnosine uptake was inhibited 94% by Gly-L-Phe, 28% by (-Ala-Gly, but not at all by Gly-D-Phe or by the amino acids -Ala and L-His. Unexpectedly, transepithelial carnosine transport at pH 7.4 (i.e., in the absence of a transepithelial pH gradient) was similar in both the apical-to-basolateral (ab) and basolateral-to-apical (ba) directions. Lowering the apical fluid pH to 6.5 reduced abtransport 1.6 times without affecting ba transport, consistent with predominantly paracellular diffusion of carnosine under an electrochemical potential gradient. Conclusions. The kinetic behavior of carnosine uptake into cultured tracheal epithelial cell layers is characteristic of a H⁺-coupled dipeptide transport process known to exist in the small intestine and the kidney. Such a process does not appear to be rate-limiting in the transport of carnosine across the tracheal epithelial barrier.     

Interaction of Nucleoside Analogues with the Sodium–Nucleoside Transport System in Brush Border Membrane Vesicles from Human Kidney

The therapeutic efficacy of nucleosides and nucleoside analogues as antitumor, antiviral, antiparasitic, and antiarrhythmic agents has been well documented. Pharmacokinetic studies suggest that many of these compounds are actively transported in the kidney. The goal of this study was to determine if therapeutically relevant nucleosides or analogues interact with the recently characterized Na⁺-driven nucleoside transport system of the brush border membrane of the human kidney. Brush border membrane vesicles (BBMV) were prepared from human kidney by divalent cation precipitation and differential centrifugation. The initial Na⁺-driven ³H-uridine uptake into vesicles was determined by rapid filtration. The effect of several naturally occurring nucleosides (cytidine, thymidine, adenosine), a pyrimidine base (uracil), a nucleotide (UMP), and several synthetic nucleoside analogues [zidovudine (AZT), cytarabine (Ara-C), and dideoxycytidine (ddC)] on Na⁺–uridine transport was determined. At a concentration of 100 µM the naturally occurring nucleosides, uracil, and UMP significantly inhibited Na+-uridine transport, whereas the three synthetic nucleoside analogues did not. Adenosine competitively inhibited Na+-uridine uptake with a K _i of 26.4 µM (determined by constructing a Dixon plot). These data suggest that naturally occurring nucleosides are substrates of the Na⁺–nucleoside transport system in the renal brush border membrane, whereas synthetic nucleoside analogues with modifications on the ribose ring are not. The K _i of adenosine is higher than clinically observed concentrations and suggests that the system may play a physiologic role in the disposition of this nucleoside.     

Transepithelial Transport of Aliphatic Carboxylic Acids Studied in Madin Darby Canine Kidney (MDCK) Cell Monolayers

Transport of ¹⁴C-labeled acetic, propionic (PA), butyric, valeric, heptanoic (HA), and octanoic (OA) acids across the Madin Darby canine kidney (MDCK) epithelial cell monolayer grown on a porous polycarbonate membrane was studied in Hanks' balanced salt solution (HBSS) at 37°C in both apical-to-basolateral and basolateral-to-apical directions. At micromolar concentrations of solutes, metabolic decomposition was significant as evidenced by [¹⁴C]CO₂ production during the OA transport. The apparent permeability (Pe) indicates that as lipophilicity increases, diffusion across the unstirred boundary layer becomes rate limiting. In support of this notion, transport of OA and HA was enhanced by agitation, showed an activation energy of 3.7 kcal/mol for OA, and resulted in identical Pe values for both transport directions. Analysis of Pe changes with varying alkyl chain length resulted in a G of –0.68 ± 0.09 kcal/mol for –CH₂-group transfer from an aqueous phase to the MDCK cells. When the intercellular tight junctions were opened by the divalent chelator EGTA in Ca²⁺/Mg²⁺ -free HBSS, transport of the fluid-phase marker Lucifer yellow greatly increased because of paracellular leakage. PA transport also showed a significant increase, but OA transport was independent of EGTA. Although albumin also undergoes paracellular transport in the presence of EGTA and OA binds strongly to albumin, OA transport in EGTA solution was unchanged by albumin. These observations indicate that transmembrane transport is the major mechanism for lipophilic substances. The present study, together with earlier work on the transport of polar substances, shows that the MDCK cell monolayer is an excellent model of the transepithelial transport barrier.     

Guanidine Transport in a Human Choriocarcinoma Cell Line (JAR)

Purpose. Many endogenous substances and xenobiotics are organic cations. Transplacental transport of organic cations is an important determinant of the delivery of these compounds to the fetus. The aim of this study was to determine the mechanisms of organic cation transport using the human choriocarcinoma cell line (JAR) as a model system with [¹⁴C]guanidine as a ligand. Methods. Uptake studies of [^l4C]guanidine were carried out in JAR cell monolayers on day 2 after plating. Results. [¹⁴C]guanidine uptake was temperature dependent, saturable (K_m = 167 M) and inhibited by many organic cations including amiloride, cimetidine, quinine, quinidine and nicotine. [^l4C]guanidine uptake exhibited a counterflux phenomenon indicative of a carrier-mediated process. The uptake of [¹⁴C]guanidine was sodium and pH-independent and could be driven by an inside-negative membrane potential difference. Conclusions. This is the first demonstration of an electrogenic guanidine transporter in a human cell culture model. This transporter may play a role in the transplacental transport of many clinically used drugs and xenobiotics.     

Transport of Clonidine at Cultured Epithelial Cells (JEG-3) of the Human Placenta

PURPOSE: This study was performed to characterize the uptake of the antihypertensive drug clonidine at epithelial cells of the human placenta (JEG-3) and its interaction with other cationic drugs. METHODS: Uptake of [3H]clonidine into cells of the human choriocarcinoma cell line JEG-3 was investigated. RESULTS: Uptake of [3H]clonidine into JEG-3 cells reached its maximum on the eighth day after seeding. Uptake of [3H]clonidine was linear for up to 1 min, Na+ independent, but strongly H+ dependent. The uptake rate of clonidine was saturable with an affinity constant (Kt) of 1.1 mM and a Vmax value of 27 nmol x min(-1) x mg(-1) of protein. Several cationic drugs such as clonidine, quinine, quinidine, and diphenhydramine strongly inhibited the [3H]clonidine uptake with Ki values around 1 mM. CONCLUSIONS: Clonidine is transported across the placental epithelium by a carrier mediated process.     

Characterization of the Amino Acid Transport of New Immortalized Choroid Plexus Epithelial Cell Lines: A Novel In Vitro System for Investigating Transport Functions at the Blood-Cerebrospinal Fluid Barrier

Purpose. To establish and characterize a choroid plexus epithelial cell line (TR-CSFB) from a new type of transgenic rat harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat).Methods. Choroid plexus epithelial cells were isolated from the Tg rat and cultured on a collagen-coated dish at 37°C during the first period of 3 days. Cells were subsequently cultured at 33°C to activate large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells using a penicillin cup.Results. Five immortalized cell lines of choroid plexus epithelial cells (TR-CSFB 15) were obtained from two Tg rats. These cell lines had a polygonal cell morphology, expressed the typical choroid plexus epithelial cell marker, transthyretin, and possessed Na⁺, K⁺-ATPase on their apical side. TR-CSFBs cells expressed a large T-antigen and grew well at 33°C with a doubling-time of 3540 hr. [³H]-L-Proline uptake by TR-CSFB cells took place in an Na⁺-dependent, ouabain-sensitive, energy-dependent, and concentration-dependent manner. It was also inhibited by -methylaminoisobutylic acid, suggesting that system A for amino acids operates in TR-CSFB cells. When [³H]-L-proline uptake was measured using the Transwell device, the L-proline uptake rate following application to the apical side was fivefold greater than that following application to the basal side. In addition, both Na⁺-dependent and Na⁺-independent L-glutamic acid (L-Glu) uptake processes were present in TR-CSFB cells.Conclusions. Immortalized choroid plexus epithelial cell lines were successfully established from Tg rats and have the properties of choroid plexus epithelial cells, and amino acid transport activity was observed in vivo.     

万古霉素对人肾小管上皮细胞中肾损伤分子-1表达的影响研究

目的:探讨万古霉素对人肾小管上皮细胞(HK-2细胞)中肾损伤分子-1(KIM-1)表达的影响及其可能机制。方法:体外培养HK-2细胞,按万古霉素作用浓度和时间分为不同浓度(0、10、20、30、40、50、60μg·mL-1)和不同时间(0、3、6、12、24、48h)组。显微镜观察HK-2细胞形态学变化,反转录-聚合酶链反应检测KIM-1mRNA表达,细胞免疫荧光法和免疫印迹法检测KIM-1蛋白表达,硫代巴比妥酸法检测HK-2细胞上清液中丙二醇(MDA)的含量。结果:镜下显示,0μg·mL-1组贴壁细胞生长良好,20、40μg·mL-1组部分贴壁细胞脱落和坏死,60μg·mL-1组大部分细胞脱落和坏死;与0μg·mL-1组比较,各浓度组KIM-1mRNA的表达均明显增强(P<0.05或P<0.01),10、20μg·mL-1组KIM-1蛋白表达明显增强(P<0.01),30、40、50、60μg·mL-1组MDA含量明显增强(P<0.01);与0h组比较,24、48h组KIM-1mRNA和蛋白表达、MDA含量均显著增强(P<0.05或P<0.01)。结论:万古霉素刺激可促进HK-2细胞KIM-1mRNA和蛋白的表达,其机制可能与万古霉素致HK-2细胞氧化应激有关。     

Role of P-Glycoprotein in Restricting Propranolol Transport in Cultured Rabbit Conjunctival Epithelial Cell Layers

Purpose. To determine the role of P-glycoprotein (P-gp) in propranololtransport in cultured rabbit conjunctival epithelial cell layers (RCEC). Methods. The localization of P-gp in the cultured RCEC as well asin the excised conjunctiva was determined by immunofluorescencetechnique. The role of P-gp in transepithelial transport and uptake ofpropranolol in conjunctival epithelial cells cultured on Transwell filterswas evaluated in the presence and absence of P-gp competing substrates, ananti-P-gp monoclonal antibody (4E3 mAb), or a metabolicinhibitor, 2, 4-dinitrophenol (2,4-DNP). Results. Immunofluorescence studies revealed positive staining in theapical membrane of cultured RCEC and in the apical surface of thesuperficial cell layers in the excised conjunctiva, but not the basolateralmembrane of cultured RCEC. Transport of propranolol showed preferencein the basolateral-to-apical direction. The net secretory flux wassaturable with a K_m of 71.5 ± 24.0 nM and a J_max of 1.45 ± 0.17pmol/cm²/hr. Cyclosporin A, progesterone, rhodamine 123, verapamil,4E3 mAb and 2,4-DNP all increased apical 50 nM propranolol uptakeby 43% to 66%. On the other hand, neither -blockers (atenolol,metoprolol, and alprenolol) nor organic cation transporter substrates(tetraethylammonium (TEA) and guanidine), affected apical 50 nMpropranolol uptake. Conclusions. The energy-dependent efflux pump P-gp appears to bepredominantly located on the apical plasma membrane of the conjunctivalepithelium. It may play an important role in restricting the conjunctivalabsorption of some lipophilic drugs.