Lowering Intracellular Calcium Concentration May Reduce the Cytotoxicity of Triamcinolone on Human Retinal Pigment Epithelial (ARPE19) Cells

1. The present study investigated the use of drugs that affect calcium (Ca^{2 +}) levels and thus reduction of triamcinolone (TA)‐induced cytotoxicity on human retinal epithelial (ARPE19) cells. 2. Four groups were compared: ARPE19 cells alone, cells exposed to TA (0.1 mg/mL), cells that have been pretreated with one of the testing agents for 30 min before the addition of TA, and cells that have only been treated with one of the testing agents. Pinacidil (PIN) and its analogue, P1060, were used to test the effect of potassium (K⁺) channel opening on TA‐induced toxicity. Verapamil (VP) and diltiazem (DZ) were used to test their Ca^{2 +} channel blocking effect. The cell viability under different settings was assessed using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Ca^{2 +}‐imaging was used to determine the changes in intracellular Ca^{2 +} levels [(Ca^{2 +})_i] upon different treatments. 3. Both PIN and P1060 reduced TA‐induced toxicity. Verapamil and DZ increased the viability of cells treated with TA significantly, suggesting that the excessive influx of Ca^{2 +} was one of the main contributory factors to the TA‐induced toxicity. 4. The results suggest that the prevention of Ca^{2 +} entry may be effective in the reduction of cell necrosis in the presence of TA.     

三氧化二砷对培养人视网膜色素上皮细胞的不良反应

目的研究三氧化二砷(As2O3)对培养的人视网膜色素上皮(human retinal pigment epithelium,HRPE)细胞的不良反应。方法培养的HRPE细胞分为血清组和无血清组,用As2O3处理HRPE细胞48h,倒置显微镜下观察细胞形态,四唑盐(MTT)比色法检测As2O3对HRPE细胞生长的影响,流式细胞仪检测As2O3处理HRPE细胞后的细胞周期变化。结果镜下观察,血清组细胞药物处理后在25μmol／L As2O3作用下出现典型凋亡细胞形态。无血清组细胞部分细胞体积增大。MTT结果显示血清组细胞3．121μmol／L As2O3时即表现出抑制作用,无血清组细胞6．25μmol／L时表现出抑制作用,流式细胞仪检测示两组细胞药物处理后细胞周期变化无区别。结论As2O3对不同生长状态下的HRPE细胞不良反应不同,可为临床玻璃体腔内用药防治增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)提供新的药物干预和实验数据。     

Expression of Multidrug Resistance-Associated Protein (MRP) in Human Retinal Pigment Epithelial Cells and Its Interaction with BAPSG,a Novel Aldose Reductase Inhibitor

Purpose. The objective of this study was to determine the expression and activity of multidrug resistance–associated protein (MRP) in the retinal pigment epithelial (RPE) cells and to further assess whether BAPSG, a novel anionic aldose reductase inhibitor, interacts with MRP. Methods. Functional and biochemical evidence for MRP was obtained in a human retinal pigment epithelial (ARPE–19) cell line and primary cultures of human retinal pigment epithelial (HRPE) cells. Fluorescein accumulation and efflux in the presence and absence of MRP inhibitors was used to obtain functional evidence for MRP. Western blots and RT–PCR were used to obtain biochemical evidence for MRP1. The influence of MRP inhibitors on BAPSG accumulation and efflux in ARPE–19 cells was determined to understand its interaction with MRP. Results. MRP inhibitors increased fluorescein accumulation and reduced efflux in RPE cells. Both cell types exhibited a 190–kDa western blot band corresponding to MRP1 protein and a 287 bp RT–PCR band corresponding to MRP1 mRNA. MRP inhibitors reduced BAPSG efflux and increased its accumulation in ARPE–19 cells. Conclusions. MRP is functionally and biochemically active in human RPE cells. Anionic BAPSG is a likely substrate for MRP.     

Effects of Tamoxifen,Toremifene and Chloroquine on the Lysosomal Enzymes in Cultured Retinal Pigment Epithelial Cells

Abstract: Retinal pigment epithelial cells carry out phagocytosis and digestion of material shed from the photoreceptor outer segments. In this process, the integrity of lysosomal enzymes is of major importance. In the present study the effects of tamoxifen, toremifene and chloroquine on the activity of two lysosomal enzymes (cathepsin D and N-acetyl-β-D-glucosaminidase) in the retinal pigment epithelial cells were studied. Retinal pigment epithelial cells from pig eyes were cultured for two weeks in Dulbecco's Modified Eagle Medium, after which the cells were exposed to 1–40 μM concentrations of tamoxifen citrate, toremifene citrate and chloroquine diphosphate. To eliminate possible medium-borne oestro-genic mechanisms, the test was repeated using phenol red-free medium with charcoal-stripped fetal calf serum. The exposure time was one week, after which the lysosomal enzymes cathepsin D and N-acetyl-β-glucosaminidase were determined. Cellular injuries were assessed by quantifying the leakage of lactate dehydrogenase into the culture medium. Cathepsin D and N-acetyl-β-D-glucosaminidase showed different sensitivities to tamoxifen, toremifene and chloroquine. The main lysosomal protease cathepsin D was more sensitive than N-acetyl-β-D-glucosaminidase to the effects of tamoxifen and toremifene, possibly due to their antioestrogenic properties. The phenol red-free medium with charcoal-stripped serum seemed to make the drugs more effective than the reference medium. Chloroquine had only a minor effect on the lysosomal protease cathepsin D, but a clearer effect could be seen on N-acetyl-β-glucosaminidase.     

神经生长因子对视网膜色素上皮细胞增殖、DNA合成及凋亡的影响

用培养的人胚胎视网膜色素上皮（Retinal pigment epithelium，RPE）细胞研究神经生长因子（Nerve growth factor，NGF）对其增殖、DNA合成及凋亡的影响。取传代培养的RPE细胞，分别用MTT（Methyl thiazolyl tetrazolium）法和核酸蛋白分析仪测定NGF对细胞增殖及DNA合成的影响；建立吲哚美辛（Indomethaein，IN）诱导细胞凋亡模型，用透射电镜（Transmission electron microsocpy，TEM）和吖啶橙（Acridine orange，AO）荧光染色研究NGF对RPE凋亡细胞的保护作用。结果表明，NGF能促进RPE细胞增殖和DNA合成，随着NGF浓度的升高而促进作用愈明显；NGF对IN诱导的RPE细胞凋亡有保护作用。     

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.     

Cellular Uptake But Low Permeation of Human Calcitonin-Derived Cell Penetrating Peptides and Tat(47-57) Through Well-Differentiated Epithelial Models

PURPOSE: To investigate whether cell penetrating peptides (CPP) derived from human calcitonin (hCT) possess, in addition to cellular uptake, the capacity to deliver their cargo through epithelial barriers. METHODS: Cellular uptake of hCT(9-32) and permeation of six hCT-derived peptides, namely, hCT(9-32), hCT(12-32), hCT(15-32), hCT(18-32), hCT(21-32), and a random sequence of hCT(9-32) were evaluated in fully organized confluent Madin-Darby canine kidney (MDCK), Calu-3, and TR146 cell culture models. For comparison, Tat(47-57) and penetratin(43-58) were investigated. The peptides were N-terminally labeled with carboxyfluorescein (CF). Uptake in the well-differentiated epithelial models was observed by confocal laser scanning microscopy (CLSM), whereas permeation through the models was analyzed by reversed-phase (RP)-HPLC. RESULTS: In MDCK epithelium hCT(9-32), Tat(47-57) and penetratin(43-58) demonstrated punctuated cytoplasmic distribution. In Calu-3, Tat(47-57) and penetratin(43-58) were simultaneously localized in a punctuated cytoplasmic and paracellular distribution, whereas hCT(9-32) showed strict paracellular distribution. By contrast, in TR146 cells, Tat(47-57) was located strictly paracellularily, whereas penetratin(43-58) showed a punctuated cytoplasmic pattern and hCT(9-32) both. The transepithelial permeability of all tested peptides and their cargo was lower than that of paracellular markers. CONCLUSIONS: The CPP uptake pattern depends on both the type of peptide and the cell culture model. In general, the investigated CPP have no apparent potential for systemic drug delivery across epithelia. Nevertheless, distinct patterns of cellular distribution may offer a potential for localized epithelial delivery.     

Comparison of Albumin Uptake in Rat Alveolar Type II and Type I-like Epithelial Cells in Primary Culture

Purpose To elucidate and compare the activity and mechanism of albumin uptake in primary cultured alveolar type II and type I-like epithelial cells. Materials and methods Type II epithelial cells isolated from rat lungs were cultured for 2 days at 5 × 10⁶ cells/35-mm dish or for 6 days at 2 × 10⁶ cells/35-mm dish. The mRNA expression of marker genes and FITC-albumin uptake were examined. Results The cells cultured for 2 days exhibited cuboidal type II epithelial morphology with lamellar bodies inside the cells, while the cells cultured for 6 days exhibited squamous type I epithelial morphology. These morphological characteristics were consistent with the changes in mRNA expression pattern of marker genes. FITC-albumin uptake in both cells was temperature-dependent and was inhibited by metabolic inhibitors and bafilomycin A₁. The rate of uptake was much higher in type II cells than type I-like cells. In both cells, FITC-albumin uptake was inhibited by clathrin mediated-endocytosis inhibitors, but not by caveolae mediated-endocytosis inhibitors. Conclusions These findings indicate that albumin in alveolar lining fluid is internalized into type II and type I epithelial cells via clathrin-mediated endocytosis, and the rate of albumin uptake is higher in type II cells than type I cells.     

Effect of Salt Forms and Molecular Weight of Chitosans on In Vitro Permeability Enhancement in Intestinal Epithelial Cells (Caco-2)

The purpose of this study was to investigate the effect of molecular weight (MW) and salt forms of chitosans (aspartate; CS A, glutamate; CS G, lactate; CS L and hydrochloride, CS HCl) on the transepithelial electrical resistance (TEER) and permeability of Caco-2 cells monolayer, using fluorescein isothiocyanate dextran 4000 (FD-4) as the model compound for paracellular tight junction transport. Chitosan salts were prepared by spray-drying method. FTIR and solid-state ¹³C NMR spectra showed the functional groups of salts in their molecular structures. Salt form, MW of chitosan, and amount of chitosan influenced the permeation-enhancing effects. These studies showed that chitosan salts appeared to increase cell permeability in a dose-dependent manner and caused relatively reversible effects only at the lower doses of 0.001–0.01% w/v. As the MW of chitosan increased from 20 to 460 kDa, the reduction in TEER significantly decreased in the following order: 20 < 45 < 200 < 460 kDa, observed in CS L and CS HCl. In CS A and CS G, the decrease in TEER was not significantly different in all MW because both chitosan salts showed rapid reduction in TEER within 20 min after the start of the experiment. Among chitosan salts, CS A was the most potent absorption enhancer in acidic (pH 6.2) environment. Cytotoxicity of chitosan salts was concentration dependent and varied slightly among the salt forms of chitosan used. CS HCl (MW 45 kDa) was the most toxic having an IC₅₀ of 0.22 ± 0.06 mg/mL. The ranking of chitosan salts cytotoxicity was CS HCl > CS L> CS G > CS A.     

Delayed Toxicity of Benzalkonium Chloride and Sodium Dodecyl Sulfate Evaluated in Primary Cultures of Rabbit Corneal Epithelial Cells

This investigation used primary cultures of rabbit corneal epithelial cells as an experimental model to evaluate the delayed ocular toxicity of benzalkonium chloride (B₂Cl) and sodium dodecyl sulfate (SDS). Initial cytotoxicity was determined after a 1-h treatment, and delayed toxicity was evaluated 2,4, and 24 h after removal of the compounds using lactate dehydrogenase (LDH) leakage into the medium and propidium iodide (PI) staining of the nucleus of the cells as markers of cellular membrane integrity. Both LDH leakage and PI staining indicated that BzCl produced minimal damage after the initial treatment but showed delayed toxicity up to 2 h after removal of the compound. There was little damage observed during the time interval of 2–24 h postexposure. In contrast, SDS caused extensive damage during initial exposure, but produced minimal increases of LDH leakage or PI staining during the postexposure periods. Reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial dehydrogenases of viable cells was determined immediately after the 1-h treatment. Twenty-four hours after the 1-h treatment with surfactants, cells were per-meabilized with digitonin, and total cellular PI staining was determined for all treatments as a relative measure of number of cells left in the culture wells. The cytotoxicity predicted from MTT reduction and total cellular PI staining was similar even though they were conducted at different times and based on different end points. We conclude that in vitro cytotoxicity assays using primary cultures of rabbit corneal epithelial cells may be used to investigate the delayed toxicity of compounds in ocular tissues. Both the lactate dehydrogenase leakage test and the propidium iodide cytotoxicity test were able to assess the detailed time-response of delayed toxicity.     

Preliminary Investigation into the Expression of Proton-Coupled Oligopeptide Transporters in Neural Retina and Retinal Pigment Epithelium (RPE): Lack of Functional Activity in RPE Plasma Membranes

Purpose. To determine the expression and functional activity of proton-coupled oligopeptide transporters (POT) in retinal pigment epithelial (RPE) cells. Methods. RT-PCR was used to probe the presence of POT mRNA in freshly isolated bovine RPE (BRPE) and human RPE (HRPE) cells, a human RPE cell line (ARPE-19), and human and bovine neural retina. [¹⁴C]GlySar uptake was used to characterize POT activity in cultured ARPE-19 cells and freshly isolated BRPE cell sheet suspensions. Results. PHT1 mRNA was expressed in BRPE, HRPE, ARPE-19, and bovine and human neural retina. In contrast, PEPT2 and PHT2 were expressed only in bovine and human retina, and PEPT1 could not be detected. GlySar exhibited a linear uptake over 6 h at pH values of 6.0 and 7.4, with greater uptake at pH 7.4 (p < 0.01). GlySar uptake did not exhibit saturability (5-2000 M) and was unchanged when studied in the presence of 1 mM L-histidine. In contrast, GlySar uptake was significantly decreased when studied at 4°C or in the presence of endocytic inhibitors at 37°C (p < 0.01). Studies in BRPE cell sheet suspensions validated the results obtained in ARPE-19 cells and strongly suggested the absence of POT on the apical and basolateral membranes of RPE. Conclusions. PHT1 mRNA is present in native bovine and human RPE and a human RPE cell line. However, the data argue against PHT1 being expressed on plasma membranes of RPE. Overall, GlySar appears to be taken up by RPE cells via a low-affinity, endocytic process.     

Evidence for LHRH-Receptor Expression in Human Airway Epithelial (Calu-3) Cells and Its Role in the Transport of an LHRH Agonist

PURPOSE: To determine whether LHRH-receptor is expressed in Calu-3, a human bronchial epithelial cell line, and to further determine whether this receptor plays a role in the transport of deslorelin, an LHRH agonist. METHODS: Using cultured monolayers of Calu-3 grown at air-interface, the presence and localization of LHRH-receptors in Calu-3 cells was determined using immunochemical methods. To determine the mechanisms of deslorelin transport, the directionality [apical-basolateral (A-B) and basolateral-apical (B-A)] of deslorelin transport across Calu-3 monolayers and the effects of temperature (37 degrees C and 4 degrees C) and an energy depletor (2,4-dinitrophenol) were investigated. To determine the role of LHRH-receptor in deslorelin transport across Calu-3 monolayers, the influence of an LHRH-receptor antisense oligonucleotide on the LHRH-receptor expression and deslorelin transport was studied. Also, the effect of a competing LHRH agonist, buserelin, on deslorelin transport was determined. RESULTS: Immunofluorescence studies indicated the predominance of LHRH-receptor in Calu-3 cells at the apical and lateral surfaces. Western blot and RT-PCR studies further confirmed the expression of LHRH-receptor in Calu-3 cells. Deslorelin transport across Calu-3 monolayers was vectorial, with the cumulative A-B transport (1.79 +/- 0.29%) at the end of 240 min being higher than the B-A transport (0.34 +/- 0.11%). Low temperature as well as 2,4-dinitrophenol abolished this directionality. LHRH-receptor antisense oligonucleotide decreased the receptor expression at the mRNA and protein level and reduced the A-B deslorelin transport by 55 +/- 4%, without affecting the B-A transport, suggesting a role for LHRH-receptor in the vectorial transport of deslorelin. In addition, buserelin reduced the A-B deslorelin transport by 56 +/- 5% without affecting the B-A transport. CONCLUSIONS: Taken together, our results provide evidence that deslorelin is transported across the respiratory epithelium via the LHRH-receptor.     

Prolonged Adverse Effects of Benzalkonium Chloride and Sodium Dodecyl Sulfate in a Primary Culture System of Rabbit Corneal Epithelial Cells

This investigation was undertaken to determine prolonged adverseeffects of benzalkonium chloride (BzCl), a cationic surfactant,and sodium dodecyl sulfate (SDS), an anionic surfactant, afteran initial treatment of and subsequent removal from a primaryculture system of rabbit corneal epithelial cells. Metabolicintegrity and cell growth were evaluated at specified periodsafter a 1-hr treatment with the surfactants because of theirimportance in tissue repair. Intracellular calcium ([Ca²⁺]₁)and intracellular pH (pH₁) were also measured because of theirimportance in cellular homeostasis. ATP/ADP ratios were usedto assess metabolic integrity, and propidium iodide stainingof cells was used to measure relative cell number and cell growth.Digitized fluorescence imaging was used to measure [Ca²⁺]₁ withfura-PE3 and pH₁ with 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein(BCECF). BzCl induced a concentration-dependent decrease inATP/ADP ratios 24 hr after its removal from the cultures, whereasSDS had minimal effects on metabolic integrity throughout the48-hr postexposure measurement period. The proliferative responseof cultures treated with SDS, however, was decreased when comparedwith BzCl-treated cells. BzCl induced an increase in [Ca²⁺]₁,whereas SDS decreased [Ca²⁺]₁ 1–3 hr after removal ofsurfactants. BzCl produced a sustained decrease in pH₁ in survivingcells 1–4 hr after its removal, with a return to controlvalues at 24–48 hr. SDS transiently increased pH₁ 1 hrafter its removal and decreased pH₁ at the 48-hr post-treatmentperiod. In conclusion, the two surfactants in vitro had distinctlydifferent prolonged effects on corneal epithelial cells, whichmay suggest that BzCl and SDS differentially affect cellularrecovery in vivo.     

Chitosans as Absorption Enhancers for Poorly Absorbable Drugs. 1: Influence of Molecular Weight and Degree of Acetylation on Drug Transport Across Human Intestinal Epithelial (Caco-2) Cells

Purpose. Chitosan has recently been demonstrated to effectively enhance the absorption of hydrophilic drugs such as peptides and proteins across nasal and intestinal epithelia (1–3). In this study, the effect of the chemical composition and molecular weight of chitosans on epithelial permeability and toxicity was investigated using monolayers of human intestinal epithelial Caco-2 cells as a model epithelium. Methods. Eight chitosans varying in degree of acetylation (DA) and molecular weight were studied. The incompletely absorbed hydrophilic marker molecule ¹⁴C-mannitol was used as a model drug to assess absorption enhancement. Changes in intracellular dehydrogenase activity and cellular morphology were used to assess toxicity. Results. Chitosans with a low DA (1 and 15%) were active as absorption enhancers at low and high molecular weights. However, these chitosans displayed a clear dose-dependent toxicity. Chitosans with DAs of 35 and 49% enhanced the transport of ¹⁴C-mannitol at high molecular weights only, with low toxicity. One chitosan (DA = 35%; MW = 170kD) was found to have especially advantageous properties such as an early onset of action, very low toxicity, and a flat dose-absorption enhancement response relationship. Conclusions. The structural features of chitosans determining absorption enhancement are not correlated with those determining toxicity, which makes it possible to select chitosans with maximal effect on absorption and minimal toxicity.     

The Effect of Cetylpyridinium Chloride (CPC) on the Cell Surface Hydrophobicity and Adherence of Candida albicansto Human Buccal Epithelial Cells in Vitro

Purpose. This study examined the effects of cetylpyridinium chloride (CPC) on cell surface hydrophobicity (CSH) and adherence of blastospores of Candida albicans(MEN strain) to human buccal epithelial cells (EEC) in vitro. Methods. The effect of CPC treatment of either C. albicans blastospores or BEC on their subsequent adherence was determined using ³⁵SO₄ labelled blastospores in association with a Percoll gradient. The effects of CPC treatment of blastospores on their CSH was determined using Hydrophobic Interaction Chromatography. Results. Treatment of exponential and stationary phase blastospores with CPC (50 µg mL^–1) for 0.5–30 minutes, or with CPC (0.5–50 µg mL^–1) for 15 minutes resulted in significant reductions in both blastospore CSH and adherence to BEC in vitro. No correlation was apparent (r < 0.8) between reduced CSH and reduced blastospore adherence following treatment with CPC (0.5–50 µg mL^–1). Significantly reduced adherence of C. albicans (stationary or exponential growth phases) to human EEC was also observed following treatment of BEC with CPC (50 µg mL^–1) for 0.5–30 minutes or with CPC (0.5–50 µg mL^–1) for 15 minutes. Antiadherence effects were observed at both sub and super-minimum inhibitory concentrations of CPC. Conclusions. It is suggested that, whilst the ability of CPC to reduce the CSH of C. albicans may contribute to its reduced adherence to human BEC in vitro, reduced CSH is only one of several possible factors that contribute to the observed antiadherence effects.     

Monolayers of Human Alveolar Epithelial Cells in Primary Culture for Pulmonary Absorption and Transport Studies

Purpose. To develop a cell culture model of human alveolar epithelial cells in primary culture for the in vitro study of pulmonary absorption and transport. Methods. Type II pneumocytes isolated from normal human distal lung tissue by enzyme treatment and subsequent purification were plated on fibronectin/collagen coated polyester filter inserts, and cultured using a low-serum growth medium. Characterization of the cell culture was achieved by bioelectric measurements, cell-specific lectin binding, immunohistochemical detection of cell junctions, and by assessment of transepithelial transport of dextrans of varying molecular weights. Results. In culture, the isolated cells spread into confluent monolayers, exhibiting peak transepithelial resistance of 2,180 ± 62 X cm² and potential difference of 13.5 ± 1.0 mV (n = 30–48), and developing tight junctions as well as desmosomes. As assessed by lectin-binding, the cell monolayers consisted of mainly type I cells with some interspersed type II cells, thus well mimicking the situation in vivo. The permeability of hydrophilic macromolecular FITC-dextrans across the cell monolayer was found to be inversely related to their molecular size, with P_app values ranging from 1.7 to 0.2 X 10^–8 cm/sec. Conclusions. A primary cell culture model of human alveolar epithelial cells has been established, which appears to be a valuable in vitro model for pulmonary drug delivery and transport studies.     

Primary Human Renal Proximal Tubular Epithelial Cells (pHRPTEpiCs): Shiga Toxin (Stx) Glycosphingolipid Receptors,Stx Susceptibility,and Interaction with Membrane Microdomains

Tubular epithelial cells of the human kidney are considered as targets of Shiga toxins (Stxs) in the Stx-mediated pathogenesis of hemolytic–uremic syndrome (HUS) caused by Stx-releasing enterohemorrhagic Escherichia coli (EHEC). Analysis of Stx-binding glycosphingolipids (GSLs) of primary human renal proximal tubular epithelial cells (pHRPTEpiCs) yielded globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Investigation of detergent-resistant membranes (DRMs) and nonDRMs, serving as equivalents for the liquid-ordered and liquid-disordered membrane phase, respectively, revealed the prevalence of Gb3Cer and Gb4Cer together with cholesterol and sphingomyelin in DRMs, suggesting lipid raft association. Stx1a and Stx2a exerted strong cellular damage with half-maximal cytotoxic doses (CD₅₀) of 1.31 × 10² pg/mL and 1.66 × 10³ pg/mL, respectively, indicating one order of magnitude higher cellular cytotoxicity of Stx1a. Surface acoustic wave (SAW) real-time interaction analysis using biosensor surfaces coated with DRM or nonDRM fractions gave stronger binding capability of Stx1a versus Stx2a that correlated with the lower cytotoxicity of Stx2a. Our study underlines the substantial role of proximal tubular epithelial cells of the human kidney being associated with the development of Stx-mediated HUS at least for Stx1a, while the impact of Stx2a remains somewhat ambiguous.