[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.

Role of microsomal glutathione transferase 1 in the mechanism-based biotransformation of glyceryl trinitrate in LLC-PK1 cells.

Although glyceryl trinitrate (GTN) has been used in the treatment of angina for many years, details of its conversion to the proximal activator (presumed to be NO or an NO congener) of soluble guanylyl cyclase (sGC) are still unclear. We reported previously that purified microsomal glutathione transferase 1 (MGST1) mediates the denitration of GTN. In the current study, we investigated in intact cells whether this enzyme also converts GTN to species that activate sGC (mechanism-based biotransformation). We utilized LLC-PK1 cells, a cell line with an intact NO/sGC/cGMP system, and generated a stable cell line that overexpressed MGST1. MGST1 in the stably transfected cells was localized to the endoplasmic reticulum, and microsomes from these cells exhibited markedly increased GST activity. Although incubation of these cells with GTN resulted in a 3-4-fold increase in GTN biotransformation, attributed primarily to an increase in formation of the 1,3-glyceryl dinitrate metabolite, GTN-induced cGMP accumulation in cells overexpressing MGST1 was not different than that observed in wild type cells or in cells stably transfected with empty vector. To determine whether overexpression of NADPH cytochrome P450 reductase might act in concert with MGST1 to generate activators of sGC, we assessed GTN-induced cGMP accumulation in MGST1-overexpressing cells that had been transiently transfected with CPR. In this case, GTN-induced cGMP accumulation was also not different than that observed in wild type cells. We conclude that although MGST1 mediates the biotransformation of GTN in intact cells, this biotransformation does not contribute to the formation of activators of sGC.

Methotrexate induces cell swelling and necrosis in renal tubular cells.

BACKGROUND: The present study was carried out to investigate if methotrexate (MTX) has a direct lethal effect in renal tubular cells, and if so, to further clarify the mechanisms of cell death. MATERIALS AND METHODS: Renal tubular cells (LLC-PK(1) cells) were incubated with MTX (0.01 microM, 0.1 microM, and 1 microM), either alone or in combination with 0.1 microM amiloride (Na(+)/H(+) antiporter inhibitor) or 1 microM carbachol (M-cholinergic agonist). Cell viability was then determined by means of trypan blue (TB) exclusion tests and MTT assays. RESULTS: After 4 hr incubation with 0.1 microM MTX the number of viable cells was decreased by 18% in comparison with control cells, and the proportion of dead cells was increased by 38%. Cell death induced by MTX was time-dependent and did not show apoptotic features. On the contrary, cell swelling was discovered. This cell death was prevented by co-incubating the cells with amiloride or carbachol. CONCLUSIONS: MTX induces cell swelling and cell death in renal tubular LLC-PK(1) cells. The tubular cell death induced by MTX is time-dependent. Cell death can be prevented by co-incubating with amiloride, thus indicating that the Na(+)/H(+) antiporter and possibly other volume regulatory factors in renal tubular cells are involved in MTX-induced renal failure.

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.

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.

Research into the development of a wearable bioartificial kidney with a continuous hemofilter and a bioartificial tubule device using tubular epithelial cells.

Current hemodialysis treatment is insufficient because of intermittent treatment and loss of tubular function. In order to overcome the loss of tubular function, a bioartificial kidney has been developed consisting of continuous hemofiltration (CHF) with 10 L/day of filtrate and a bioartificial tubule device using proximal tubular epithelial cells and hollow fiber membranes. Ten L/day of CHF enabled plasma levels of urea, creatinine, uric acid and, beta2-microglobulin in eight renal failure patients to be maintained at remarkably low levels. The concept was tested with 6 L (4 mL/min) of 10 L/day (7 mL/min) filtrate regenerated by a bioartificial tubule device and 4 L/day (3 mL/min) replaced by food and drinks. Lewis lung cancer-porcine kidney 1 (LLC-PK1) cells with a cell density of 107 cells/mL were seeded inside polysulfone hollow fiber modules four times at 1 h intervals while rotating the module 90 degrees each time, and were cultured for 48 h to form confluent monolayers. The leak rates of urea and creatinine across LLC-PK1 cell-attached polysulfone membrane modules (membrane areas: 56 cm2 and 4000 cm2) were investigated. Via conversion from 56 m2 to 1 m2 hollow fiber modules with LLC-PK1 cells for 24 h, the transport rates of H2O, glucose and Na+ were, respectively, 40, 65 and 35% of the target transported amounts from 6 L/day of filtrate. The rates are expected to approach 100% when 4-5 g/dL of albumin is added to the basal portion of the medium since the results were obtained without the addition of albumin for colloidal osmotic pressure.

Studies of cell division (mitosis and cytokinesis) by dynamic secondary ion mass spectrometry ion microscopy: LLC-PK1 epithelial cells as a model for subcellular isotopic imaging.

The feasibility of the renal epithelial LLC-PK1 cell line as a model for cell division studies with secondary ion mass spectrometry (SIMS) was tested. In this cell line, cells undergoing all stages of mitosis and cytokinesis remained firmly attached to the substrate and could be cryogenically prepared. Fractured freeze-dried mitotic cells showed well-preserved organelles as revealed by fluorescence imaging of rhodamine-123 and C6-NBD-ceramide by confocal laser scanning microscopy. Secondary electron microscopy analysis of fractured freeze-dried dividing cells revealed minimal surface topography that does not interfere in isotopic imaging of both positive (39K, 23Na, 24Mg, 40Ca, etc.) and negative (31P, 35Cl, etc.) secondaries with a CAMECA IMS-3f ion microscope. Mitotic cells revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of 39K+ and 23Na+) as revealed by K : Na ratios of approximately 10. Structurally damaged mitotic cells could be identified by their reduced K : Na ratios and an excessive loading of calcium. Quantitative three-dimensional SIMS analysis was required for studying subcellular calcium distribution in dividing cells. The LLC-PK1 model also allowed SIMS studies of M-phase arrested cells with mitosis-arresting drugs (taxol, monastrol and nocodazole). This study opens new avenues of cell division research related to ion fluxes and chemical composition with SIMS.

Role of polymeric Tamm-Horsfall protein in cast formation: oligosaccharide and tubular fluid ions.

BACKGROUND: In acute tubular necrosis (ATN), distal tubules are obstructed by casts formed by tubular debris, cells, and Tamm-Horsfall protein (THP). Since there are Arginine-Glycine-Aspartate (RGD) and Leucine-Aspartate-Valine (LDV) adhesive sequences in human THP, there may be direct integrin-mediated binding of tubular cells to THP. Alternatively, polymerization of THP may result in entrapment of the cells in its gel. METHODS: Adhesion of LLC-PK(1) cells to THP-coated wells was directly measured. THP concentrate was dissolved in solutions which mimic urine from ATN (ATN-S), distal convoluted tubule (DCT-S), collecting duct (CD-S), and monomeric buffer (M buffer). THP was also denatured by either boiling or N-glycanase digestion. Gel formation of THP was then measured. Inhibition of LLC-PK(1) cell adhesion to collagen type I was measured with each solution, as well as after the collagen was pretreated with either monomeric (mTHP) or polymeric (pTHP) THP. The effect of pTHP on the settling rate of LLC-PK(1) cells in suspension was also measured. RESULTS: LLC-PK(1) cells did not directly adhere to THP, a finding against integrin-mediated binding as a mechanism for in vivo tubular cell/THP cast formation. The high electrolyte concentration of ATN-S and CD-S, however, was associated with pTHP gel formation. Moreover, cells suspended in pTHP remained in suspension. In cell adhesion studies, mTHP attenuated cell adhesion by binding to the matrix, whereas pTHP attenuated cell adhesion by trapping cells in its gel. An active process was involved since both pTHP gel formation and attenuation of cell adhesion were abolished by boiling or oligosaccharide removal with N-glycanase digestion. CONCLUSIONS: With renal ischemia and proximal tubule cell shedding, ATN and collecting duct fluid composition enhance THP gel formation and thus favor tubular cast formation and obstruction. The present in vitro results indicate the importance of oligosaccharide residues in mediating the effect of the THP on gel formation and potential cast formation in ATN.

Aristolochic acid I-induced apoptosis in LLC-PK1 cells and amelioration of the apoptotic damage by calcium antagonist.

OBJECTIVE: To examine the effect of different concentrations of aristolochic acid I (AAI) in inducing apoptosis of cultured porcine renal cell line LLC-PK1 and to investigate the relationship between intracellular free calcium concentration ([Ca++]i) and LLC-PK1 apoptosis induced by AAI and the influence of a calcium antagonist, lacidipine on apoptosis and [Ca++]i. METHODS: LLC-PK1 cells were treated in different groups: a. the normal group without treatment; b. the group with AAl alone (0.01 g.L-1, 0.02 g.L-1, 0.04 g.L-1, 0.08 g.L-1); c. the group with lacidipine alone (10 ng.L-1, 10(2) ng.L-1, 10(3) ng.L-1); d. the group with AAI (0.04 g.L-1) plus lacidipine (10 ng.L-1, 10(2) ng.L-1, 10(3) ng.L-1). Light microscopy, agarose gel electrophoresis, Annexin-V-Flous apoptosis detection kit and flow cytometry using propidium iodide staining to identify or quantify the apoptosis of LLC-PK1 cells. Mean [Ca++]i was measured by laser confocus microscopy using Fluo-3/AM staining. RESULTS: A series of morphologic changes that were characteristic of apoptosis, Annexin-V-Flous staining positive apoptotic cells and "DNA ladder" were identified in AAI (0.02 g.L-1-0.08 g.L-1) treated LLC-PK1 cells. Quantitative analysis of apoptotic cells showed that the percentage of apoptotic cells in AAI (0.02 g.L-1, 0.04 g.L-1 or 0.08 g.L-1) group was significantly higher than that in normal group (5.3%, 48.5%, 78.7% vs 2.6%, P < 0.001). Mean [Ca++]i was significantly higher in cells treated with AAI (0.04 g.L-1) than that in normal cells (58.01 +/- 18.89 vs 22.66 +/- 4.78, P < 0.001). In group treated with AAI plus lacidipine (102 ng.L-1, 103 ng.L-1), mean [Ca++]i was significantly lower than that treated with AAI alone (35.47 +/- 12.85, 28.55 +/- 10.16 vs 58.01 +/- 18.89, P < 0.001). And the percentage of apoptotic cells in group treated with AAI plus lacidipine (10(2) ng.L-1, 10(3) ng.L-1) was also significantly lower than that treated with AAI alone (19.0%, 27.8% vs 34.7%, P < 0.001). CONCLUSIONS: High concentrations of AAI may induce apoptosis of LLC-PK1 cells. The mean [Ca++]i in AAI-treated LLC-PK1 cells was increased significantly, suggesting that the increase of [Ca++]i may be related to apoptosis in LLC-PK1 cells. Lacidipine may decrease the raised mean [Ca++]i levels caused by AAI and the percentage of apoptotic cells, and lacidipine may ameliorate AAI-induced apoptotic damage by inhibiting the increase of [Ca++]i in LLC-PK1 cells.

Effects of glibenclamide on glycylsarcosine transport by the rat peptide transporters PEPT1 and PEPT2.

1 Glibenclamide is a widely used sulphonylurea for the treatment of non-insulin-dependent diabetes mellitus (NIDDM). This agent has been reported to inhibit the activities of various ion channels and transporters. In the present study, we examined the effects of glibenclamide on the function of the H+/peptide cotransporters PEPT1 and PEPT2 by using stable transfectants. 2 Uptake of [14C]-glycylsarcosine, a typical substrate for peptide transporters, by PEPT1- or PEPT2-expressing transfectant was inhibited by glibenclamide as well as other sulphonylureas including tolbutamide. 3 Kinetic analysis revealed that the inhibition by glibenclamide was noncompetitive. Dixon plot analyses showed that the Ki values of this agent were 25 and 7.8 microM for PEPT1 and PEPT2, respectively. 4 Glibenclamide did not inhibit Na+-coupled alanine and alpha-methyl-D-glucoside transport, suggesting that the inhibitory effects of glibenclamide on peptide transporters were not due to nonspecific interactions. 5 There was little uptake of [3H]-glibenclamide by PEPT-expressing transfectants as compared to mock-transfected cells, suggesting that glibenclamide was not a substrate for these peptide transporters. 6 In summary, glibenclamide inhibited the [14C]-glycylsarcosine transport by PEPT1 and PEPT2 in a noncompetitive fashion, although glibenclamide per se was not transported through these transporters. These findings would provide important information for clinical, physiological and biochemical aspects of peptide transporters.

Cyclosporine A-induced cell cycle arrest and cell death in renal epithelial cells.

We studied the effects of cyclosporine A (CsA) on the proliferation of LLC-PK1 proximal tubule epithelial cells. DNA damage was found to be an early event in CsA nephrotoxicity and could be a sensitive indicator of CsA injury in renal epithelial cells. Cell cycle arrest induced by CsA was coincident with elevated p53 levels. It is possible that trans-activating p21 may mediate the halting of the cell cycle through the CsA-induced accumulation of p53.

Apoptosis induced by cisplatin nephrotoxic injury.

An agent often used in cancer treatment, cisplatin may cause acute renal failure after even a single dose. Recent laboratory studies confirmed the induction of renal cell apoptosis by cisplatin. Reduction in cell viability as well as increase in the number of cells with fragmented nuclei correlated with cisplatin exposure in a dose-dependent manner. Gel electrophoresis revealed the presence of a characteristic laddering pattern which was preceded by an increase in caspase-3 activity. As the cisplatin concentration increased beyond 50 microM, the elevation of caspase-3 activity declined, which suggests that necrosis, instead of apoptosis, is more likely to be responsible for cell death secondary to higher cisplatin concentrations. Elucidation of the molecular mechanisms of cisplatin nephrotoxicity may lead to the development of a novel therapeutic strategy that targets cancer cell death while simultaneously minimizing renal injury.

Inhibition of cell differentiation by G alpha q in the renal epithelial cell line LLC-PK1.

LLC-PK1, an epithelial cell line derived from the kidney proximal tubule, was used to study the ability of the G protein alpha-subunit, G alpha q, to regulate cell differentiation. A constitutively active mutant protein, alpha qQ209L, was expressed using the LacSwitch-inducible mammalian expression system. Induction of alpha qQ209L expression with isopropyl-beta-D-thiogalactopyranoside (IPTG) enhanced phospholipase C activity maximally by 6- to 7.5-fold. Increasing concentrations of IPTG progressively inhibited the activity of two differentiation markers, Na(+)-dependent hexose transport and alkaline phosphatase activity. Induction of alpha qQ209L expression also caused a change from an epithelial to a spindle-shaped morphology. The effects of alpha qQ209L expression on cell differentiation were similar to those observed with 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment. However, protein kinase C (PKC) levels were downregulated in TPA-treated cells but not in alpha qQ209L-expressing cells, suggesting that the regulation of PKC by G alpha q may be different from regulation by TPA. Interestingly, the PKC inhibitor GF-109203X did not inhibit the effect of IPTG on the development of Na(+)-dependent hexose transport in alpha qQ209L-expressing cells. These data implicate PKC delta and PKC epsilon in the pathway used by G alpha q to block the development of Na(+)-dependent hexose transport in IPTG-treated cells.

Aminoglycoside antibiotics traffic to the Golgi complex in LLC-PK1 cells.

Aminoglycoside antibiotics are known to be internalized via endocytosis and have been associated with subcellular organelle dysfunction; however, the route of intracellular trafficking and their distribution remain largely unknown. To address these questions, a Texas Red conjugate of gentamicin (TRG) was synthesized for dual-labeling experiments with the endoplasmic reticulum, Golgi, and lysosomal markers DiOC6-3, C6-NBD-ceramide, and fluorescent dextrans, respectively. Confocal images were overlaid to determine areas of colocalization. Initial characterization studies of the fluorescent gentamicin analogue revealed that both internalization and accumulation were inhibited by excess unlabeled gentamicin. Furthermore, the fluorescent gentamicin label was colocalized with unlabeled gentamicin, using immunologic techniques. LLC-PK1 cells were exposed to the fluorescent gentamicin in media containing 1 mg/ml labeled gentamicin for 8 h and then either fixed or chased with gentamicin-free media for an additional 16 or 40 h (24 to 48 h total). Studies with fluorescent dextrans revealed rapid intracellular colocalization within the endosomal and lysosomal systems. Neither endoplasmic reticulum nor mitochondrial colocalization could be detected. However, Golgi colocalization was revealed using both confocal and electron microscopic techniques at 8 h of TRG incubation, and continued to be present for an additional 40 h. Protein synthetic rates were quantified and revealed decreased synthesis at the 24-h chase mark. These results suggest that TRG can serve as a fluorescent tracer for aminoglycoside trafficking within cells. The fluorescent marker remained associated with vesicular structures at all times and colocalized with the Golgi apparatus. It is postulated that this early association of gentamicin with the Golgi complex may be an avenue for delivery of aminoglycosides to other intracellular compartments.

Renal accumulation and excretion of cyclic adenosine monophosphate in a murine model of slowly progressive polycystic kidney disease.

Evidence from in vitro studies indicates that increased proliferation of epithelial cells and secretion of fluid by these cells may be important factors in the progressive enlargement of renal cysts. The rate of cellular proliferation and fluid secretion by cyst epithelium in vitro can be strikingly accelerated by cyclic adenosine 3'5' monophosphate (cAMP) and agonists that lead to the production of this nucleotide. The extent to which renal cAMP content is increased in polycystic kidneys is unknown. In the current study, we determined the amount of this nucleotide in intact kidneys, cyst fluid, plasma, and urine in nonazotemic mice (DBA/2FG-pcy/pcy) with a slowly progressive form of inherited polycystic kidney disease (PKD). In 45 pcy/pcy mice studied 20, 45, or 70 days after birth, the total kidney cAMP content was 0.22 +/- 0.01, 0.46 +/- 0.02, and 0.90 +/- 0.05 pmol/mg tissue, respectively. By contrast, in 37 control DBA/2J mice the levels of cAMP at identical times remained relatively constant at 0.22 +/- 0.01, 0.21 +/- 0.01, and 0.29 +/- 0.01 pmol/mg tissue, respectively. In 70-day-old nonazotemic pcy/pcy mice with normal serum levels of parathyroid hormone, cAMP generated by the kidneys (nephrogenous cAMP) was 22.9 +/- 2.8 nmol/100 mL creatinine clearance, compared with 6.5 +/- 1.3 in normal animals of the same age (P < 0.001). The cyst fluids of 70-day-old pcy/pcy mice contained a lipid that increased transepithelial secretion of fluid by MDCK monolayers from a baseline of 0.012 +/- 0.002 to 0.136 +/- 0.008 microL/cm2/hr (P < 0.05). This lipid also stimulated cellular proliferation by monolayers of cultured MDCK and LLC-PK1 cells 2.5- and 7.9-fold (P < .05), respectively, and stimulated cAMP accumulation by these cells 1.6- and 2.0-fold (P < .05), respectively. These studies illustrate that renal cAMP production and excretion increase in concert with the cystic enlargement of the kidneys in DBA/2FG-pcy/pcy mice and identify a lipid cAMP agonist in murine renal cystic kidney disease.

Role of caspases (ICE/CED 3 proteases) in DNA damage and cell death in response to a mitochondrial inhibitor, antimycin A.

Caspases (ICE/ Ced3 proteases) are a closely related family of cysteine proteases that play a key role in apoptotic cell death. We examined the role of caspases in DNA damage and cell death in response to the mitochondrial inhibitor, antimycin A. LLC-PK1 cells contain caspase activity that was markedly inhibited by cleavage site-based peptide inhibitors of caspases but not by inhibitors of serine, cysteine, aspartate or metalloproteinases. The caspase activity increased within five minutes of exposure to antimycin A, preceding any evidence of DNA damage and cell death. The specific caspase inhibitors. Ac-Tyr-Val-Ala-Asp-aldehyde (inhibitor I) and Ac-Asp-Glu-Val-Asp-aldehyde (inhibitor II) prevented, in a dose dependent manner, antimycin A-induced DNA strand breaks as determined by DNA unwinding assay (residual double stranded DNA in control, 94 +/- 2%; antimycin A alone, 48 +/- 3%; antimycin A + inhibitor I at 50 microM, 93 +/- 2%; antimycin A + inhibitor II at 50 microM, 89 +/- 5%; N = 3 to 4, P < 0.001). These inhibitors also prevented antimycin A-induced DNA fragmentation as determined by agarose gel electrophoresis and by in situ labeling of cell nuclei by the terminal deoxynucleotidyl transferase (TdT) nick end labeling (TUNEL) method. The caspase inhibitors markedly prevented antimycin A-induced cell death in a dose-dependent manner as measured by trypan blue exclusion (control 6 +/- 1%, antimycin A alone 40 +/- 1%, antimycin A + inhibitor I at 50 microM 16 +/- 1%, antimycin A + inhibitor II at 50 microM 16 +/- 1%; N = 4 to 7, P < 0.001). These data indicate that the caspase family of enzymes play an important role in DNA damage and cell death in response to the mitochondrial inhibitor, antimycin A.

Ezrin is an effector of hepatocyte growth factor-mediated migration and morphogenesis in epithelial cells.

The dissociation, migration, and remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) entail modifications in cell adhesion and in the actin cytoskeleton through unknown mechanisms. Here we report that ezrin, a membrane-cytoskeleton linker, is crucial to HGF-mediated morphogenesis in a polarized kidney-derived epithelial cell line, LLC-PK1. Ezrin is a substrate for the tyrosine kinase HGF receptor both in vitro and in vivo. HGF stimulation causes enrichment of ezrin recovered in the detergent-insoluble cytoskeleton fraction. Overproduction of wild-type ezrin, by stable transfection in LLC-PK1 cells, enhances cell migration and tubulogenesis induced by HGF stimulation. Overproduction of a truncated variant of ezrin causes mislocalization of endogenous ezrin from microvilli into lateral surfaces. This is concomitant with altered cell shape, characterized by loss of microvilli and cell flattening. Moreover, the truncated variant of ezrin impairs the morphogenic and motogenic response to HGF, thus suggesting a dominant-negative mechanism of action. Site-directed mutagenesis of ezrin codons Y145 and Y353 to phenylalanine does not affect the localization of ezrin at microvilli, but perturbs the motogenic and morphogenic responses to HGF. These results provide evidence that ezrin displays activities that can control cell shape and signaling.

Different size limitations for increased transepithelial paracellular solute flux across phorbol ester and tumor necrosis factor-treated epithelial cell sheets.

By observing increases in the transepithelial paracellular permeability of a range of radiolabeled solutes and electron dense dyes, changes in molecular sieving caused by the cytokine, TNF (tumor necrosis factor), and the phorbol ester, TPA (12-0-tetra-decanoylphorbol-13-acetate), were characterized. Using 14C-labeled mannitol (mw 182), raffinose (mw 504), PEG (polyethylene glycol; mw 4000), and dextran (mw 10,000, 70,000 and 2,000,000), the transepithelial flux rates of these compounds were determined at the peak of the transepithelial electrical resistance (TER) changes caused by these two agents. TNF treatment resulted in increased permeability across LLC-PK1 epithelial cell sheets only to relatively small solutes, with an upper limit of approximately 4,000 mw. The low molecular weight "ceiling" for the TNF-treated epithelium is further evidence against TNF increasing transepithelial permeability by means of inducing nonspecific, microscopic "holes" in the epithelium, for which a "ceiling" would not exist. TPA treatment increases transepithelial paracellular permeability to a much broader range of solutes, extending well beyond 2 million mw. Transmission electron micrographs provide evidence that even the electron-dense dye complex, ruthenium red, can cross tight junctions of TPA-treated cell sheets. However, cationic ferritin cannot cross tight junctions of TPA-treated cell sheets. This shows that there is an upper limit to solutes able to cross TPA-treated cell sheets, but that this upper limit will include most proteins, which would then be able to cross tumor promoter-exposed (protein kinase C-activated) epithelial layers at accelerated rates. The biomedical implications for a high molecular weight cutoff in tumor promoter action in epithelial carcinogenesis, and for a low molecular weight cutoff in cytokine-induced epithelial apoptosis in inflammation, are discussed.

Tolerance to cadmium cytotoxicity is induced by zinc through non-metallothionein mechanisms as well as metallothionein induction in cultured cells.

To investigate the relationship between metallothionein induction and zinc-induced tolerance to cadmium cytotoxicity at the cell level, Chang liver cells were pretreated with a wide range of concentrations of zinc sulfate and challenged with toxic levels of cadmium after removal of zinc. It was found that zinc-pretreated cells were significantly resistant even when the extracellular level of zinc was low and metallothionein was not induced by the metal. Pretreatment with zinc resulted in a lesser accumulation of cadmium and metallothionein after challenge with cadmium. In porcine kidney LLC-PK1 cells and bovine aortic smooth muscle cells pretreated with low levels of zinc, the intracellular accumulation of cadmium was significantly less and cadmium-induced decrease in intracellular zinc was significantly prevented without a change of metallothionein content; as the result, the toxic effect of cadmium was significantly diminished. It was therefore concluded that a tolerance to cadmium cytotoxicity is induced by zinc via non-metallothionein mechanisms such as decreasing intracellular cadmium as well as metallothionein induction at the cell level. The non-metallothionein mechanism was postulated to be particularly importatnt when the extracellular concentration of zinc is under the threshold for metallothionein induction.

Effects of high glucose culture on EGF effects and EGF receptors in the LLC-PK1 cells.

We have shown that epidermal growth factor (EGF) may be important in diabetic renal hypertrophy. Since EGF is the most potent mitogen for the proximal tubule, it may be relevant to the cellular hyperplasia component in diabetic nephropathy. In order to further clarify the possible alterations of mitogenic effects of EGF on cultured renal cells in hyperglycemic states, the effects of high glucose culture on EGF-induced events and EGF receptors were studied in LLC-PK1 cells with equimolar mannitol being used as an osmotic control. The results showed that high glucose dose-dependently decreased mitogenesis while increasing cellular hypertrophy in LLC-PK1 cells. The dose-response curve of EGF-induced mitogenesis was similar in both normal (11 mM) and high (27.5 mM) glucose cultures. Meanwhile, EGF receptor number and affinity were not changed by high glucose in these cells. Furthermore, mannitol mimicked the growth-suppressive (but not hypertrophic) effects of high glucose cultures. Based upon these findings, we conclude that high glucose did not alter the mitogenic effects of EGF on the LLC-PK1 cells. This was associated with unchanged EGF receptor characteristics. Thus, concurrent with our previous studies, we speculate that it is the increased local EGF level, rather than an increased renal sensitivity to it, which is associated with hyperglycemic tubulopathy.