Elevated glucose concentration in culture media decreases membrane trafficking of SGLT2 in LLC-PK1 cells via a cAMP/PKA-dependent pathway.

Na+-dependent glucose reabsorption in the renal proximal tubule is dynamically regulated by changes in blood glucose levels. There is, however, a disparity in reports studying the relationship between hyperglycemia and Na+-glucose-linked transporter (SGLT) function and expression. Similarly, manipulation of the glucose content in growth media of cultured renal cells has been shown to influence SGLT activity. In this investigation, SGLT activity was significantly lower in proximal tubule LLC-PK1 cells cultured in medium containing 17.5 than 5 mM glucose. α-Methyl d-glucopyranoside (AMG) transport kinetics showed reduced apparent Vmax and Km in cells grown in 17.5 mM glucose. SGLT2 was identified as the isoform responsible for glucose transport, and protein expression analyses showed decreased apical membrane localization of SGLT2 in cells grown in 17.5 mM glucose, explaining the reduced activity. Multiple signaling pathways have been implicated in regulation of SGLT activity and trafficking. Elevated media glucose decreased intracellular cAMP and PKA activation, leading to decreased SGLT2 trafficking into the plasma membrane, which was reversed after treatment with 1 µM forskolin. The effects of media glucose on SGLT activity were found to be dependent on p38 MAPK activation due to PKA-mediated signaling. Glucose-modulated AMG uptake is reversible and was associated with altered SGLT2 membrane trafficking and cAMP alterations. In summary, elevated glucose concentrations in culture medium decrease SGLT activity in LLC-PK1 cells by reducing membrane trafficking of SGLT2 via decreasing intracellular cAMP, resulting in a lowered PKA-dependent phosphorylation of p38 MAPK.

Effect of Danofloxacin on Reactive Oxygen Species Production, Lipid Peroxidation and Antioxidant Enzyme Activities in Kidney Tubular Epithelial Cell Line, LLC-PK1.

The purpose of this study was to investigate the possibility that oxidative stress was involved in danofloxacin-induced toxicity in renal tubular cells epithelial cell line (LLC-PK1). Confluent LLC-PK1 cells were incubated with various concentrations of danofloxacin. The extent of oxidative damage was assessed by measuring the reactive oxygen species (ROS) level, lipid peroxidation, cell apoptosis and antioxidative enzyme activities. Danofloxacin induced a concentration-dependent increase in the ROS production, not even cytotoxic conditions. Similarly, danofloxacin caused an about 4 times increase in the level of thiobarbituric acid reactive substances at the concentration of 400 µM for 24 hr, but it did not induce cytotoxicity and apoptosis. Antioxidant enzymes activities, such as superoxide dismutase (SOD) and catalase (CAT), were increased after treatment with 100, 200 and 400 µM of danofloxacin for 24 hr. The activity of glutathione peroxidase (GPX) was significantly decreased in a concentration-dependent manner. In addition, ROS production, lipid peroxidation and GPX decline were inhibited by additional glutathione and N-acetyl cysteine. These data suggested that danofloxacin could not induce oxidative stress in LLC-PK1 cells at the concentration (≤400 µM) for 24 hr. The increase levels of ROS and lipid peroxidation could be partly abated by the increase activities of SOD and CAT.

[Establishment of BCRP expressed pig kidney cell line LLC-PK1/BCRP and its biological profile].

To establish a pig kidney cell line LLC-PK1/BCRP in which human breast cancer resistance protein was highly expressed, the expression vector pcDNA3.1(+)-BCRP which contained BCRP gene was constructed and transfected into LLC-PKI cells via liposomes. After selecting with G418, population doubling time, flow cytometry and Western blotting analysis were used to evaluate the cell line. MTT assays were employed to determine the drug resistance index of mitoxantrone and doxorubicin. Invert fluorescent microscope was used to observe the efflux of fluorescence dye Hoechst 33342 by BCRP, furthermore, the BCRP's inhibitor GF120918 was applied to reverse the efflux of Hoechst 33342. The experiment results showed that the expression of BCRP protein increased in LLC-PK1/BCRP cell. The population doubling time of LLC-PK1/BCRP cell was a little longer than that of the parental cell LLC-PK1. The resistance indexes to mitoxantrone and doxorubicin were 51.95 and 6.09 times, respectively, higher than LLC-PK1 cell. The efflux of Hoechst 33342 was significantly enhanced and could be reversed by GF120918. So a LLC-PK1/BCRP cell line was established, which highly expressed BCRP protein successfully. This cell line could be a valuable model to further investigate the biological profile of BCRP and select the substrate and inhibitor of BCRP.

Protective effect of sulforaphane against cisplatin-induced mitochondrial alterations and impairment in the activity of NAD(P)H: quinone oxidoreductase 1 and γ glutamyl cysteine ligase: studies in mitochondria isolated from rat kidney and in LLC-PK1 cells.

This work was designed to further study the mechanism by which sulforaphane (SFN) exerts a renoprotective effect against cisplatin (CIS)-induced damage. It was evaluated whether SFN attenuates the CIS-induced mitochondrial alterations and the impairment in the activity of the cytoprotective enzymes NAD(P)H: quinone oxidoreductase 1 (NQO1) and γ glutamyl cysteine ligase (γGCL). Studies were performed in renal epithelial LLC-PK1 cells and in isolated renal mitochondria from CIS, SFN or CIS+SFN treated rats. SFN effectively prevented the CIS-induced increase in reactive oxygen species (ROS) production and the decrease in NQO1 and γGCL activities and in glutathione (GSH) content. The protective effect of SFN on ROS production and cell viability was prevented by buthionine sulfoximine (BSO), an inhibitor of γGCL, and by dicoumarol, an inhibitor of NQO1. SFN was also able to prevent the CIS-induced mitochondrial alterations both in LLC-PK1 cells (loss of membrane potential) and in isolated mitochondria (inhibition of mitochondrial calcium uptake, release of cytochrome c, and decrease in GSH content, aconitase activity, adenosine triphosphate (ATP) content and oxygen consumption). It is concluded that the protection exerted by SFN on mitochondrial alterations and NQO1 and γGCL enzymes may be involved in the renoprotection of SFN against CIS.

The protection of selenium on ROS mediated-apoptosis by mitochondria dysfunction in cadmium-induced LLC-PK(1) cells.

Selenium, an essential trace element, showed the significant protective effects against liver and kidney damage induced by some heavy metals. However, the mechanism how selenium suppresses cadmium (Cd)-induced cytotoxicity remains unclear. In this study, we investigated the protective mechanism of selenium on Cd-induced apoptosis in LLC-PK(1) cells via reactive oxygen species (ROS) and mitochondria linked signal pathway. Studies of PI and Annexin V dual staining analysis demonstrated that 20 microM Cd-induced apoptosis as early as 18 h. A concomitant by the generation of ROS, the loss of mitochondrial membrane potential, cytochrome c (cyt c) release, activation of caspase-9, -3 and regulation of Bcl-2 and Bax were observed. N-acetylcysteine (NAC, 500 microM), a free radical scavenger, was used to determine the involvement of ROS in Cd-induced apoptosis. During the process, selenium played the same role as NAC. The anti-apoptosis exerted by selenium involved the blocking of Cd-induced ROS generation, the inhibition of Cd-induced mitochondrial membrane potential collapse, the prevention of cyt c release, subsequent inhibition of caspase activation and the changed level of Bcl-2 and Bax. Taken together, we concluded that Cd-induced apoptosis was mediated by oxidative stress and selenium produced a significant protection against Cd-induced apoptosis in LLC-PK(1) via ameliorating the mitochondrial dysfunction.

Aristolochic acid I-induced DNA damage and cell cycle arrest in renal tubular epithelial cells in vitro.

DNA damage is a critical event preceding cellular apoptosis or necrosis. This study was carried out to investigate the effect of aristolochic acid I (AAI) on DNA damage and cell cycle in porcine proximal tubular epithelial cell lines (LLC-PK1 cells). LLC-PK1 cells were stimulated with AAI at the concentrations of 80, 320, and 1,280 ng/ml for 24 h. DNA damage was examined by comet assay and the cell cycle was assayed by flow cytometry (FCM), cellular apoptosis and lysis were examined simultaneously. Cellular nuclear changes were observed by electron microscopy and the expression of wild-type p53 protein and mRNA were measured by FCM and RT-PCR. We found that AAI-induced DNA damage prior to apoptosis and lysis in LLC-PK1 cells in a dose-dependent manner (P<0.01). The percentage of cells in the G2/M phase that were treated with AAI (320 and 1,280 ng/ml) for 24 h increased significantly (P<0.01). Electron micrographs showed the nuclear abnormalities in AAI-treated cells. The expression of p53 protein and mRNA did not change in the AAI-treated cells. AAI may cause DNA damage and cell cycle arrest in LLC-PK1 cells through a wild-type p53-independent pathway, prior to apoptosis or necrosis. This study on the molecular mechanism of AAI-induced toxicity may explain why tubular epithelial cells present limited proliferation and regeneration abilities in the clinical presentation of AAI-associated nephrotoxicity.

Evaluation of proliferation and functional differentiation of LLC-PK1 cells on porous polymer membranes for the development of a bioartificial renal tubule device.

To develop a bioartificial renal tubule system using renal tubular cells and porous polymer membrane hollow fibers, long-term maintenance of a confluent monolayer and the functionally differentiated condition of cells is essential. We examined the proliferation and functional differentiation of LLC-PK1 (Lewis-lung cancer porcine kidney 1) cells on two types of membranes: polysulfone and cellulose acetate. Cell proliferation was significantly higher on the polysulfone membrane than on the cellulose acetate membrane, and was enhanced by coating the membranes with various extracellular matrices. Confluent monolayer formation of cells was observed on matrix-coated polysulfone membrane but not on matrix-coated cellulose acetate membrane within 1 week. Cell proliferation continued for 3 weeks after confluent monolayer formation. Messenger RNA (mRNA) expression of glucose transporters, indicators of the functional differentiation of the LLC-PK1 cells, was observed in the polysulfone and cellulose acetate membrane groups, but was not observed in the nonporous polystyrene plate group under subconfluent conditions. Expression of glucose transporters mRNA was maintained for 3 weeks after confluent monolayer formation. Polysulfone membrane is more suitable than cellulose acetate membrane for a bioartificial renal tubule system with regard to LLC-PK1 cell proliferation. Extracellular matrix coating of the membrane further improves cell proliferation.

Interaction of N1,N12-diacetylspermine with polyamine transport systems of polarized porcine renal cell line LLC-PK1.

LLC-PK(1) cells grown on porous membrane filters were employed as a model system to explore the renal transport of polyamines. The polarity of LLC-PK(1) monolayers was confirmed by the exclusive appearance of a Na(+)-dependent alpha-methylglucoside transport system on the apical surface. The uptake of free polyamines from the basolateral side of monolayers was consistent with the existence of a single class of transport system, while the existence of two kinetically distinct polyamine transport systems with higher and lower affinities on apical membranes was suggested. The results of competition studies indicated that each of these transporters was able to interact with putrescine, spermidine and spermine. LLC-PK(1) cells incorporated monoacetylspermine from the apical surface of monolayers at about half the rate of spermine uptake. Monoacetylspermine inhibited spermidine uptake, indicating that free polyamine transport systems also recognized the monoacetylated derivative. In contrast, N(1),N(12)-diacetylspermine did not inhibit spermidine uptake, nor was it incorporated into the cells, indicating the absence of transport systems that recognize N(1),N(12)-diacetylspermine on the apical membranes of LLC-PK(1) cells. These results may be relevant as to our previous observation that the content of diacetylpolyamines in urine is relatively constant, and may explain the excellence of N(1),N(12)-diacetylspermine as a tumor marker.

O papel da heme oxigenase-1 na lesão celular desencadeada pelo sulfato de polimixina B em células LLC-PK1 / The role of heme oxygenase-1 in the cellular injury caused by polymyxin B sulphate on LLC-PK1 cells

A heme-oxigenase-1 (HO-1) é uma enzima induzível envolvida na degradação do grupo prostético heme, produzindo compostos com funções anti-oxidante, anti-inflamatória, anti-apoptótica e modulatória do sistema imune no rim. A importância de sua indução está associada à resposta adaptativa ao estresse oxidativo e à inflamação envolvidos na gênese da insuficiência renal aguda. O sulfato de polimixina B é um antibiótico usado no tratamento de infecções Gram-negativas e que apresenta um efeito nefrotóxico ainda não completamente elucidado. O objetivo deste estudo foi verificar a viabilidade e apoptose de células LLC-PK1 submetidas ao tratamento com polimixina B, com tempos de exposição diferentes, e pré-tratadas com hemin (indutor de heme oxigenase-1) ou protoporfirina de zinco (inibidor de heme oxigenase-1). Células renais de porco, LLC-PK1, foram cultivadas com polimixina B durante 24, 48 e 72 horas. A apoptose e viabilidade celular foram avaliadas usando diferentes doses do antibiótico: Controle (CTL, 0 µM); G1 (12,5µM); G2 (37,5µM); G3 (75µM); G4 (125µM) e G5 (375µM). O hemin (25µM) e a protoporfirina de zinco (10µM) foram administrados uma hora antes da polimixina B. Foram utilizados os métodos Acridine orange/ brometo de etídio (viabilidade) e Hoescht 33342 (apoptose). Os resultados demonstraram redução linear de viabilidade induzida pela polimixina B quando a dose e o tempo de exposição foram aumentados, isto foi confirmado pela variação inversa de apoptose. O hemin aumentou a viabilidade e reduziu apoptose na presença de polimixina B, sugerindo um efeito protetor da HO-1 neste modelo. O efeito observado para a protoporfirina de zinco foi semelhante ao descrito para o hemin. O estudo confirmou a citotoxicidade da polimixina B em células renais e constatou que esse efeito pode ser mediado pela HO-1 considerando o efeito obtido no tratamento com o indutor daquela enzima

Functional role of the NPxxY motif in internalization of the type 2 vasopressin receptor in LLC-PK1 cells.

Interaction of the type 2 vasopressin receptor (V2R) with hormone causes desensitization and internalization. To study the role of the V2R NPxxY motif (which is involved in the clathrin-mediated endocytosis of several other receptors) in this process, we expressed FLAG-tagged wild-type V2R and a Y325F mutant V2R in LLC-PK1a epithelial cells that have low levels of endogenous V2R. Both proteins had a similar apical (35%) and basolateral (65%) membrane distribution. Substitution of Tyr325 with Phe325 prevented ligand-induced internalization of V2R determined by [3H]AVP binding and immunofluorescence but did not prevent ligand binding or signal transduction via adenylyl cyclase. Desensitization and resensitization of the V2R-Y325F mutation occurred independently of internalization. The involvement of clathrin in V2R downregulation was also shown by immunogold electron microscopy. We conclude that the NPxxY motif of the V2R is critically involved in receptor downregulation via clathrin-mediated internalization. However, this motif is not essential for the apical/basolateral sorting and polarized distribution of the V2R in LLC-PK1a cells or for adenylyl cyclase-mediated signal transduction.

Cyclosporin A disrupts bradykinin signaling through superoxide.

Cyclosporin A (CsA) is used to reduce transplant rejection rates. Chronic use, however, has a destructive toxic effect on the kidney, resulting in hypertension. In this study, we investigated the effects of CsA treatment on the bradykinin/soluble guanylate cyclase signaling cascade and the involvement of superoxide in LLC-PK1 porcine kidney proximal tubule cells. Treatment with 1 micromol/L CsA for 24 hours increased basal cGMP levels by 41%, whereas CsA inhibited bradykinin-stimulated cGMP production by 26%. Western blotting showed increased expression of eNOS, but no other protein in the bradykinin/soluble guanylate cyclase (sGC) pathway was affected. Using lucigenin-dependent chemiluminescence, we found that CsA treatment significantly increased superoxide production. Production of O2- was not significantly reduced by 10 micromol/L oxypurinol or 30 micromol/L ketoconazole. However, it was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium chloride (10 micromol/L) as well as the O2- scavenger superoxide dismutase (SOD) (100 U). On treatment with 50 micromol/L quercetin, 10 mmol/L N-acetyl-cysteine, both antioxidants, as well as the O2- scavenger Tiron (10 mmol/L), concomitant with 1 micromol/L CsA for 24 hours the activation of cGMP production, was restored in combination with a reduction in O2-. Incubation with 100 micromol/L menadione, a reactive oxygen generator, and 10 nmol/L bradykinin showed similar effects on the level of cGMP as with CsA. CsA treatment was found to increase nitrotyrosine levels. These findings suggest that CsA activates a NADPH oxidase that releases O2- and disrupts the bradykinin/soluble guanylate cyclase pathway, probably by binding with NO to form peroxynitrite (ONOO-).

Up-regulation of MDR1 function and expression by cisplatin in LLC-PK1 cells.

To examine whether cisplatin affects the multidrug transporter MDR1/P-glycoprotein in the kidneys, the effects of cisplatin on cell sensitivity to an anticancer drug, MDR1 function and expression were examined by assessing the growth inhibition by the MDR1 substrate paclitaxel, the uptake and efflux of the MDR1 substrate Rhodamine123 and the level of MDR1 mRNA, respectively. Porcine kidney epithelial LLC-PK1 cells were used, as they have a structure and function similar to those of renal proximal tubular cells and physiologically express low levels of MDR1. The growth inhibitory curve of LLC-PK1 cells by paclitaxel was shifted to a higher concentration range by pretreatment with 1 micro M cisplatin for 48 h. The uptake and efflux of Rhodamine123 were significantly reduced and enhanced, respectively, by pretreatment with 1 micro M cisplatin for 48 h. This enhanced efflux was suppressed by the representative MDR1 substrate/inhibitor ciclosporin. The expression of MDR1 mRNA was increased by the existence of cisplatin for 48 h. These observations taken together suggested that the transient exposure to cisplatin could cause the up-regulation of MDR1 in LLC-PK1 cells.

Analysis of methotrexate and folate transport by multidrug resistance protein 4 (ABCC4): MRP4 is a component of the methotrexate efflux system.

Human MRP4 (ABCC4, MOAT-B) is a lipophilic anion transporter that is able to confer resistance to nucleotide analogues and methotrexate (MTX). We previously investigated the implications of the ability of MRP4 to confer resistance to nucleotide analogues and determined that the pump is competent in the MgATP-energized transport of cyclic nucleotides and estradiol 17beta-D-glucuronide. Here we examine the potential role of MRP4 in conferring resistance to MTX and related processes by determining the selectivity of the transporter for MTX, MTX polyglutamates, and physiological folates. In so doing, it is shown that MRP4 is active in the transport of MTX as well as the physiological folates folic acid (FA) and N(5)-formyltetrahydrofolic acid (leucovorin). MTX, FA, and leucovorin are subject to high capacity [V(max(MTX)), 0.24 +/- 0.05 nmol/mg/min; V(max (FA)), 0.68 +/- 0.14 nmol/mg/min; V(max(leucovorin)), 1.95 +/- 0.18 nmol/mg/min], low affinity [K(m(MTX)), 0.22 +/- 0.01 mM; K(m(FA)), 0.17 +/- 0.02 mM; K(m (leucovorin)), 0.64 +/- 0.23 mM] transport by MRP4. In addition, as would be expected were MRP4 a component of the MTX efflux system, its capacity to transport this agent is abrogated by the addition of a single glutamyl residue. It is also shown that glutamylation similarly affects the ability of MRP2 to transport MTX. On the basis of these transport properties, it is concluded that the efflux system for MTX includes MRP2 and MRP4, in addition to MRP1 and MRP3, and that MRP4 represents a common efflux system for both MTX and certain nucleotide analogues.

Induction of uPA but not NF-IL3A by calcitonin is dependent on Erk1/2 phosphorylation in porcine renal cell line LLC-PK1.

Calcitonin (CT) is a polypeptide hormone and has a variety of functions including regulation of urinary calcium excretion. By using a cDNA subtraction hybridization method, we identified that NF-IL3A and urokinase-type plasminogen activator (uPA) genes were up-regulated by CT in porcine renal cell line LLC-PK1. CT-mediated induction of these genes was not inhibited by cycloheximide. These data suggest that these up-regulations are not induced by increased synthesis of regulating proteins; therefore, they are immediately response early (IE). We also found that CT treatment led to the phosphorylation of Erk1/2. We demonstrated that PD98059, a MEK1 inhibitor, inhibited CT-induced mRNA expressions of uPA, but had no obvious influence on the NF-IL3A induction. These results demonstrated the inductions of uPA by CT involve Erk1/2 phosphorylation. We provide the first evidence that NF-IL3A expression is up-regulated by CT. The present findings suggest that the transcriptions of the NF-IL3A and uPA could be induced by CT and might be important mediators of CT function in renal cells.

Effect of 17 beta-estradiol-D-17 beta-glucuronide on the rat organic anion transporting polypeptide 2-mediated transport differs depending on substrates.

Rat organic anion transporting polypeptide 2 (rOatp2) is a member of the OATP family. It exhibits broad substrate specificity and accepts amphipathic organic anions, cardiac glycosides (digoxin and ouabain; a neutral compound), and organic cations (rocuronium and N-(4,4-azo-n-pentyl)-21-deoxyajamalinium). In the present study, kinetic analyses were carried out to investigate whether taurocholate (TCA), digoxin, and 17beta-estradiol-D-17beta-glucuronide (E(2)17betaG) share the same recognition site on rOatp2 for their transport. The transport of TCA and digoxin was mutually inhibited, and the K(i) values of digoxin and TCA for the transport of TCA and digoxin were 0.58 and 160 microM, respectively. The K(m) and V(max) values of TCA and digoxin were 190 microM and 140 pmol/min/mg of protein and 1.1 microM and 6.6 pmol/min/mg of protein, respectively. The K(m) and K(i) values were consistent. In addition, digoxin (1 microM) and TCA (100 microM) increased the K(m) values of TCA and digoxin, respectively, but they did not affect the V(max) values, suggesting that their inhibition is competitive. The transport of digoxin via rOatp2 was inhibited slightly by E(2)17betaG, whereas the uptake of TCA was stimulated by E(2)17betaG in a concentration-dependent manner. These results suggest that rOatp2 has at least two substrate recognition sites, one for TCA and digoxin and the other for E(2)17betaG.

Species- and sex-specific renal cytotoxicity of ochratoxin A and B in vitro.

Four different cell models were chosen for comparison of OTA and OTB toxicity: primary porcine (PKC), rat (RPTC) and human renal proximal epithelial cells (HKC) from both sexes and a porcine renal cell line: LLC-PK1. Culture conditions were tested and optimized for each respective cell type (species/sex and origin). All cell types were characterized for epithelial origin and growth patterns and following optimization of dosing strategies and assay procedures, a strict study design was implemented to avoid systemic variations. Due to possible sensitivity differences, three simple endpoints were chosen to provide basic data for interspecies comparison: neutral red uptake, MTT reduction and cell number. Of the endpoints tested neutral red appeared the most sensitive, although all three parameters yielded comparable EC50's. Sex-differences were observed between male and female HKC cells following 96 h exposure to OTA, with HKC(m) being more sensitive than HKC(f). No sex-difference was observed in PKC cells, however, the PKC were approximately 3 and 10 times more sensitive than HKC(m) and HKC(f), respectively, to OTA and OTB. Interestingly, the CI95 of the EC50 values obtained for OTA (15.5-16.5 microM) and OTB (17.0-2 1.0 microM) were comparable in the PKC cells. In contrast, OTB had lower cytotoxicity than OTA in HKC and LLC-PK1 (approx. 2-fold) and no effects in RPTC. Overall, HKC(m) were nearly as sensitive as PKC towards OTA, followed by RPTC, LLC-PK1 and HKC(f), thus suggesting a sex specific sensitivity in humans towards OTA induced cytotoxicity.

Inhibitory effects of CYP3A4 substrates and their metabolites on P-glycoprotein-mediated transport.

It is generally known that the substrates and/or inhibitors of cytochrome P450 (CYP) 3A4 and P-glycoprotein (P-gp) overlap with each other. In intestinal epithelial cells, it is surmised that the metabolites coexist with their parent drug. However, most studies on P-gp did not take the effects of those metabolites into consideration. Therefore, in the present study, we investigated the inhibitory effects of five substrates of CYP3A4 (nifedipine, testosterone, midazolam, amiodarone, and azelastine) and their metabolites on the P-gp-mediated transcellular transport. The transcellular transports of [(3)H]daunorubicin or [(3)H]digoxin by monolayers of LLC-GA5-COL150 cells in which P-gp was overexpressed were measured in the presence or absence of the CYP3A4 substrates and their metabolites. Nifedipine, testosterone, midazolam, and their metabolites exhibited no effects on the P-gp-mediated transport of [(3)H]daunorubicin and [(3)H]digoxin. On the other hand, the transport of [(3)H]daunorubicin was strongly inhibited by amiodarone, desethylamiodarone, azelastine, and desmethylazelastine, with IC(50) values of 22.5, 15.4, 16.0 and 11.8 microM, respectively. The transport of [(3)H]digoxin was also strongly inhibited by these compounds, with IC(50) values of 45.6, 25.2, 30.0 and 41.8 microM, respectively. Another metabolite of azelastine, 6-hydroxyazelastine, exhibited no effects on these transports. It was suggested that the CYP3A4 metabolites of which their parent drug exhibited inhibition on the P-gp-mediated transport are possibly also inhibitors. It would be possible more complicated drug-drug interactions would be caused by the metabolites as well as their parent drugs in the liver and the intestine via the inhibition of CYP3A4 and P-gp.

Effect of cyclosporin A on nitric oxide production in cultured LLC-PK1 cells.

The effect of Cyclosporin A on nitric oxide production was studied in cultured LLC- PK1 cells. For this purpose the cells were incubated with vehicle (olive oil, 10 microg/ml in DMSO), Cyclosporin A (CsA, 10 microg/ml), tumor necrosis factor (TNF-alpha, 150 U/ml) + interferon (IFN-gamma, 500 U/ml) to upregulate NOS synthesis, and therefore NO production (used as a positive control), or CsA + TNF-alpha + IFN-gamma. After 72 hours the culture medium was collected and nitrite was determined by the Griess method. The results were normalized to the protein harvested from these cells as measured by the Lowry method. Viability was determined by the exclusion of the fluorescent dyes (acridine orange and ethidium bromide). Intracellular calcium was measured spectrophotometrically using the fluorescent calcium indicator fura-2 AM. In CsA treated cells, the nitrite (pmoles/mg of protein) was decreased when compared to control (12.8 +/- 0.5 vs. 18.3 +/- 0.6; p < 0.05; both n = 8). TNF-alpha + IFN-gamma increased the nitrite synthesis (52.0 +/- 0.2; p < 0.05 vs. control; n = 6). This effect was decreased significantly by the simultaneous treatment with CsA (38.8 +/- 0.3; p < 0.05; n = 6). Cell viability in CsA group was decreased when compared to the control (84.7 +/- 0.2% vs. 93.6 +/- 0.1%; p < 0.05; both n = 10). TNF-alpha + IFN-gamma had no effect on viability (93.0 +/- 0.3%; n = 10). However, when combined with CsA, viability was decreased relative to the control (85.0 +/- 0.2%; p < 0.05; n = 10). Acute (1 h) or chronic (72 h) treatment of LLC- PK1 cells with CsA had no effect on basal calcium levels. Our results demonstrate a reduced level of nitric oxide production in LLC-PK1 cells treated with CsA. There was no effect of the drug on intracellular calcium levels, however CsA treatment did reduce cellular viability. We suggest that, in part, the decreased levels of NO production are a secondary consequence of direct cell damage. However, CsA may also be exerting direct effects on NO synthesis through its interactions with both iNOS and cNOS. These results also provide a dual mechanism of action for CsA induced nephrotoxicity, that is, direct cell damage and interference with the NO system within the nephron.

The Delta F508 mutation shortens the biochemical half-life of plasma membrane CFTR in polarized epithelial cells.

Although the biosynthetic arrest of the DeltaF508 mutant of cystic fibrosis transmembrane conductance regulator (CFTR) can be partially reversed by physical and chemical means, recent evidence suggests that the functional stability of the mutant protein after reaching the cell surface is compromised. To understand the molecular basis for this observation, the current study directly measured the half-life of Delta F508 and wild-type CFTR at the cell surface of transfected LLC-PK(1) cells. Plasma membrane CFTR expression over time was characterized biochemically and functionally in these polarized epithelial cells. Surface biotinylation, streptavidin extraction, and quantitative immunoblot analysis determined the biochemical half-life of plasma membrane DeltaF508 CFTR to be approximately 4 h, whereas the plasma membrane half-life of wild-type CFTR exceeded 48 h. This difference in biochemical stability correlated with CFTR-mediated transport function. These findings indicate that the Delta F508 mutation decreases the biochemical stability of CFTR at the cell surface. We conclude that the Delta F508 mutation triggers more rapid internalization of CFTR and/or its preferential sorting to a pathway of rapid degradation.

Cytofluorimetric analysis of a renal tubular cell line and its resistant counterpart.

P-glycoprotein (P-gp) and multidrug resistance related protein (MRP) overexpression is often responsible of the development of multidrug resistance in cancer therapy. These proteins are also expressed in normal tissues, where their physiological role is related to the extrusion of endogenous toxins or to secretory function in liver and kidney. The LLC-PK1 cell line is derived from normal pig proximal renal tubule and physiologically expresses low levels of P-gp and MRP. A resistant cell line (LLC-PK1/ADR) has been established in our laboratory by chronic exposure to increasing doses of doxorubicin. Cytofluorimetric analysis of P-gp and MRP expression performed by C219 and MRPm6 immunofluorescence detection showed that these cells overexpress P-gp but not MRP. The uptake of doxorubicin and rhodamine 123 has been quantified in LLC-PK1 and LLC-PK1/ADR cells and compared with data obtained using other tumor cell lines commonly used as reference for studying P-gp or MRP overexpression. P388 sensitive cells and its resistant counterpart P388/ADR cells, which overexpress P-gp and PANC-1 cells, which express high levels of MRP were used. A lower fluorescence intensity was evident with both doxorubicin and rhodamine 123 in LLC-PK1/ADR as well as in P388/ADR cells, that overexpresses P-gp, in comparison with the parental lines. The uptake was increased by a pretreatment with verapamil. Verapamil was completely ineffective on PANC-1 cells, confirming a selective effect of this inhibitor on P-gp. Propidium iodide staining, performed after doxorubicin treatment, confirmed a higher cytotoxicity of the antineoplastic drug in the LLC-PK1 cells compared with the resistant counterpart.