Rat Alveolar Epithelial Cell Monolayers Research Articles

Biokinetic Modeling of Nanoparticle Interactions with Lung Alveolar Epithelial Cells

Many studies on the health effects of inhaled ambient and engineered nanomaterials (ENMs) have provided information on nanoparticle toxicity and translocation across airway and alveolar epithelial barriers. Various inhaled ENMs (e.g., gold and iridium nanoparticles) have been reported to partially cross the air-blood barrier in the lung, enter the circulation and distribute into several end organs, including heart, liver, spleen and kidney. Using an in vitro primary rat alveolar epithelial cell (AEC) monolayer model, we reported transport rates of relatively nontoxic polystyrene nanoparticles (PNPs), which appear to be taken up via nonendocytic processes into AECs. PNPs internalized into cytoplasm then trigger autophagy, followed by delivery of PNPs from autophagosomes into lysosomes, from where PNPs can be exocytosed. We used the data from these in vitro experiments to perform biokinetic modeling that incorporates the processes associated with internalization and intracellular distribution of PNPs, autophagy, lysosomal exocytosis of PNPs, and several putative mechanisms of action that extend our previous understanding of AEC processing of PNPs. Results indicate that entry of PNPs into AECs, subsequent activation of autophagy by cytosolic PNPs, accumulation of PNPs in lysosomes, and lysosomal exocytosis are interwoven by complex proposed regulatory mechanisms. These findings are consistent with the presence of intracellular interactions that provide potential new insights into how inhaled nanoparticles may be mechanistically linked to the development of acute and chronic lung injury.

Characteristics of Passive Solute Transport across Primary Rat Alveolar Epithelial Cell Monolayers.

Primary rat alveolar epithelial cell monolayers (RAECM) were grown without (type I cell-like phenotype, RAECM-I) or with (type II cell-like phenotype, RAECM-II) keratinocyte growth factor to assess passive transport of 11 hydrophilic solutes. We estimated apparent permeability (Papp) in the absence/presence of calcium chelator EGTA to determine the effects of perturbing tight junctions on “equivalent” pores. Papp across RAECM-I and -II in the absence of EGTA are similar and decrease as solute size increases. We modeled Papp of the hydrophilic solutes across RAECM-I/-II as taking place via heterogeneous populations of equivalent pores comprised of small (0.41/0.32 nm radius) and large (9.88/11.56 nm radius) pores, respectively. Total equivalent pore area is dominated by small equivalent pores (99.92–99.97%). The number of small and large equivalent pores in RAECM-I was 8.55 and 1.29 times greater, respectively, than those in RAECM-II. With EGTA, the large pore radius in RAECM-I/-II increased by 1.58/4.34 times and the small equivalent pore radius increased by 1.84/1.90 times, respectively. These results indicate that passive diffusion of hydrophilic solutes across an alveolar epithelium occurs via small and large equivalent pores, reflecting interactions of transmembrane proteins expressed in intercellular tight junctions of alveolar epithelial cells.

Biokinetic modeling of nanoparticle interactions with lung alveolar epithelial cells: uptake, intracellular processing, and egress.

Studies on health effects of engineered nanomaterials (ENMs) in the lung have provided information on ENM toxicity and translocation across airway and alveolar epithelial barriers. Various inhaled ENMs (e.g., gold and iridium nanoparticles) have been reported to partially cross the air-blood barrier in the lung, enter the vasculature, and distribute in several end organs, including the heart, liver, spleen, and kidney. Using an in vitro primary rat alveolar epithelial cell (AEC) monolayer model, we reported transport rates of relatively nontoxic polystyrene nanoparticles (PNPs), which appear to be taken up via nonendocytic processes into AECs. PNPs internalized into cytoplasm then trigger autophagy, followed by delivery of PNPs from autophagosomes into lysosomes, from where PNPs are exocytosed. We used the data from these experiments to perform biokinetic modeling that incorporates the processes associated with internalization and intracellular distribution of PNPs, autophagy, lysosomal exocytosis of PNPs, and several putative mechanisms of action that extend our previous understanding of AEC processing of PNPs. Results suggest that entry of PNPs into AECs, subsequent activation of autophagy by cytosolic PNPs, accumulation of PNPs in lysosomes, and lysosomal exocytosis are interwoven by proposed regulatory mechanisms.

Evidence for Nanoparticle-Induced Lysosomal Dysfunction in Lung Adenocarcinoma (A549) Cells.

Background: Polystyrene nanoparticles (PNP) are taken up by primary rat alveolar epithelial cell monolayers (RAECM) in a time-, dose-, and size-dependent manner without involving endocytosis. Internalized PNP in RAECM activate autophagy, are delivered to lysosomes, and undergo [Ca2+]-dependent exocytosis. In this study, we explored nanoparticle (NP) interactions with A549 cells. Methods: After exposure to PNP or ambient pollution particles (PM0.2), live single A549 cells were studied using confocal laser scanning microscopy. PNP uptake and egress were investigated and activation of autophagy was confirmed by immunolabeling with LC3-II and LC3-GFP transduction/colocalization with PNP. Mitochondrial membrane potential, mitophagy, and lysosomal membrane permeability (LMP) were assessed in the presence/absence of apical nanoparticle (NP) exposure. Results: PNP uptake into A549 cells decreased in the presence of cytochalasin D, an inhibitor of macropinocytosis. PNP egress was not affected by increased cytosolic [Ca2+]. Autophagy activation was indicated by increased LC3 expression and LC3-GFP colocalization with PNP. Increased LMP was observed following PNP or PM0.2 exposure. Mitochondrial membrane potential was unchanged and mitophagy was not detected after NP exposure. Conclusions: Interactions between NP and A549 cells involve complex cellular processes leading to lysosomal dysfunction, which may provide opportunities for improved nanoparticle-based therapeutic approaches to lung cancer management.

Interactions of Inhaled Nanoparticles with Rat Alveolar Epithelial Cell Monolayers

RationaleTo understand the contribution to lung pathogenesis of ambient ultrafine air pollution particles, knowledge of basic mechanisms underlying how inhaled nanoparticles interact with alveolar epithelial cells (AEC) is needed. We have reported that polystyrene nanoparticles (PNP) at the apical surface of primary rat alveolar AEC monolayers (RAECM) are taken up in a manner dependent on exposure time, dose and particle size via a non‐endocytic ‘diffusional’ process, induce autophagy, and exit via both fast intracellular calcium‐dependent lysosomal exocytosis and a slower (‘diffusional’) process. In this study, we further investigated intracellular fate of PNP.MethodsRAECM were apically exposed to near‐infrared labeled PNP (80 μg/mL; carboxylated; 20 nm). AEC were subjected to confocal imaging throughout the volume of the cells. In each z plane, total intracellular fluorescence intensity as well as vesicular and cytosolic PNP were determined and normalized to total intracellular PNP (PNPic). Volumes of endosomal, autophagosomal and lysosomal compartments, and colocalization of PNP in cytosolic vs vesicular compartments, were assessed using an early endosome marker (Rab5) tagged with green fluorescent protein (GFP), quinacrine, microtubule‐associated protein 1 light chain 3 (LC3B)‐GFP, and Magic Red (a lysosomal marker). [PNPic] was then determined by an isotropic method (cuvette‐based microfluorometry) utilizing AEC lysates as a function of time. [PNP] in vesicles ([PNPv]) and cytosol ([PNPc]) were estimated from the respective fractions of compartmental volumes and co‐localized PNP in each compartment.ResultsAfter 12 hr, [PNPic] reached a plateau of 1.84 mg/mL. Distribution of intracellular PNP was ~80% in intracellular vesicles (primarily lysosomes) and ~20% in cytosol at steady state, equivalent to 8.86 mg/ml and 0.58 mg/mL for [PNPv] and [PNPc], respectively. About 3% of total intracellular PNP was observed in Rab5‐GFP positive early endosomes by 3 hr after PNP exposure, but inhibition of classical endocytosis pathways did not alter PNP uptake kinetics. In contrast, blockade of autophagosome formation by 3‐methyladenine (3MA) markedly reduced PNP uptake and decreased steady state [PNPic] by ~75% with no detectable vesicular PNP. Inhibition of autophagosome‐lysosome fusion by bafilomycin lowered steady state PNPic by ~20% compared to control, with ~81% in cytosol and ~19% in vesicles.SummaryIn AEC exposed to PNP, [PNPv] exceeds both [PNPc] and [PNPic], with [PNPc]>>apical [PNP]. Cytosolic presence of PNP triggers autophagosome formation, leading to PNP accumulation in lysosomes. Inhibition of autophagy leads to decreased PNP entry and [PNPic]. These data suggest that intracellular processing of PNP is regulable, offering possible approaches to amelioration of inhaled nanoparticle‐related cytotoxicity in AEC.Support or Funding InformationFunding: NIH; Will Rogers Institute; Hastings and Whittier FoundationsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oligopeptide Transport in Rat Lung Alveolar Epithelial Cells is Mediated by Pept2.

Studies were conducted in primary cultured rat alveolar epithelial cell monolayers to characterize peptide transporter expression and function. Freshly isolated rat lung alveolar epithelial cells were purified and cultured on permeable support with and without keratinocyte growth factor (KGF). Messenger RNA and protein expression of Pept1 and Pept2 in alveolar epithelial type I- and type II-like cell monolayers (±KGF, resp.) were examined by RT-PCR and Western blotting. 3H-Glycyl-sarcosine (3H-gly-sar) transmonolayer flux and intracellular accumulation were evaluated in both cell types. RT-PCR showed expression of Pept2, but not Pept1, mRNA in both cell types. Western blot analysis revealed presence of Pept2 protein in type II-like cells, and less in type I-like cells. Bi-directional transmonolayer 3H-gly-sar flux lacked asymmetry in transport in both types of cells. Uptake of 3H-gly-sar from apical fluid of type II-like cells was 7-fold greater than that from basolateral fluid, while no significant differences were observed from apical vs. basolateral fluid of type I-like cells. This study confirms the absence of Pept1 from rat lung alveolar epithelium in vitro. Functional Pept2 expression in type II-like cell monolayers suggests its involvement in oligopeptide lung disposition, and offers rationale for therapeutic development of di/tripeptides, peptidomimetics employing pulmonary drug delivery.

Effects of nanoparticle size and surface charge on uptake into rat alveolar epithelial cells

Physicochemical properties of nanoparticles, including size, composition and surface charge, are likely to affect nanoparticle interactions with biological cells/tissues. We reported previously that uptake of polystyrene nanoparticles (PNP) from apical fluid into primary cultured rat alveolar epithelial cell monolayers (RAECM) takes place in a manner dependent on exposure time and dose. Classical endocytosis pathways do not appear to play a role in the uptake of PNP, consistent with a ‘diffusional’ process. We also reported that autophagy is involved in cellular processing of PNP, in that accumulation of PNP in lysosomes occurs via autophagic flux. Lysosomal exocytosis of PNP (modulated by intracellular [Ca2+]), takes place in parallel with a slower ‘diffusional’ egress process. In this study, we investigated the effects of nanoparticle size and surface charge on uptake into RAECM grown on Anopore filters. RAECM were exposed apically to 80 μg/mL carboxylated or amidinated 20, 100 or 200 nm PNP (with near‐infrared fluorescent label) and intracellular [PNP] was monitored over 24 hr using live cell imaging. Results showed that intracellular [PNP] reached a plateau of ~0.6 mg/mL after ≥12 hr of apical exposure to both 20 nm carboxylated or amidinated [PNP] at 80 μg/mL. As PNP size increased from 20 to 100 to 200 nm, steady state intracellular [PNP] decreased from ~0.6 to ~0.3 to ~0.1 μg/mL, respectively, after ≥12 hr. These data indicate that, for RAECM, PNP uptake is strongly dependent on PNP size, while the role of surface charge appears to be less important. Additional factors (e.g., nanoparticle‐associated protein corona) might alter the charge effect. In general, understanding the role of physicochemical properties of nanoparticles in cellular interactions will lead to improved nanoparticle design for protection from cellular injury and approaches to therapeutic utilization in drug/gene delivery.Support or Funding InformationFunding: NIH; Hastings and Whittier Foundations.

Superoxide mediates tight junction complex dissociation in cyclically stretched lung slices

We found that stretching Type I rat alveolar epithelial cell (RAEC) monolayers at magnitudes that correspond to high tidal-volume mechanical ventilation results in the production of reactive oxygen species, including nitric oxide and superoxide. Scavenging superoxide with Tiron eliminated the stretch-induced increase in cell monolayer permeability, and similar results were reported for rats ventilated at large tidal volumes, suggesting that oxidative stress plays an important role in barrier impairment in ventilator-induced lung injury associated with large stretch and tidal volumes. In this communication we show that mechanisms that involve oxidative injury are also present in a novel precision cut lung slices (PCLS) model under identical mechanical loads. PCLSs from healthy rats were stretched cyclically to 37% change in surface area for 1 hour. Superoxide was visualized using MitoSOX. To evaluate functional relationships, in separate stretch studies superoxide was scavenged using Tiron or mito-Tempo. PCLS and RAEC permeability was assessed as tight junction (TJ) protein (occludin, claudin-4 and claudin-7) dissociation from zona occludins-1 (ZO-1) via co-immunoprecipitation and Western blot, after 1h (PCLS) or 10min (RAEC) of stretch. Superoxide was increased significantly in PCLS, and Tiron and mito-Tempo dramatically attenuated the response, preventing claudin-4 and claudin-7 dissociation from ZO-1. Using a novel PCLS model for ventilator-induced lung injury studies, we have shown that uniform, biaxial, cyclic stretch generates ROS in the slices, and that superoxide scavenging that can protect the lung tissue under stretch conditions. We conclude that PCLS offer a valuable platform for investigating antioxidant treatments to prevent ventilation-induced lung injury.

Effect of surfactants on polystyrene nanoparticle (PNP) interactions with primary rat alveolar epithelial cell monolayers (RAECM)

We investigated effects of apical 0.1% (w/v) Pluronic (P) or 1 mg/mL Infasurf (I) on flux of 20 nm carboxyl‐modified (negatively charged) PNP (20‐PNP) or 20 nm amidine‐modified (positively charged) PNP (20+ PNP) across RAECM, across which the bioelectric properties (transepithelial resistance (Rt) and equivalent short circuit current (Ieq)) were unchanged in the presence of these two surfactants. Results of flux measurements, along with hydrodynamic diameter (HD) measured by dynamic light scattering and zeta potential (ZP) measured in the presence and absence of P or I, are summarized below. 20+ PNP 20‐ PNP Flux (pg/cm2/sec) PNP alone 38±7 1.6±0.1 PNP with P 35±7 2.5±0.2 PNP with I 42±6 0.9±0.2 HD (nm) PNP alone 543±28 156±7 PNP with P 374±13 80±3 PNP with I 316±9 278±9 ZP (mV) PNP alone 37±10 −61±6 PNP with P 33±2 −29±2 PNP with I −44±1 −82±4 These data indicate that there is remarkably little effect of P or I on PNP flux despite significant changes in measured HD and ZP. We conclude that PNP (and RAECM) exposure to either surfactant P or I surprisingly does not cause meaningful alterations in PNP translocation across RAECM, which may be explainable by the behavior of PNP in the extracellular microdomain adjacent to the apical alveolar epithelial cell membrane.(Funded by NIH and Hastings & Whittier Fdns.)

Nanodiamond (ND) interactions with primary rat alveolar epithelial cell monolayers (RAECM)

We investigated interactions of positively and negatively charged 5 nm ND (zeta potentials of 50 and −60 mV, respectively; provided by Carbodeon, Vantaa, Finland) with RAECM. Transepithelial resistance (Rt) and equivalent short circuit current (Ieq) of RAECM in the presence and absence of ND in apical fluid for up to 24 h did not change. We estimated cellular uptake and transepithelial trafficking of ND across RAECM over 24h spectrofluorometrically with excitation/emission wavelengths at 275/555 nm. Cell uptake of positively charged ND was ~3 times greater than that (~9 pg/μg cell protein) of negatively charged ND, while transepithelial trafficking of positively and negatively charged ND was about the same at ~20 pg/cm2/s. Trafficking of ND is ~10‐fold faster than that of negatively or positively charged quantum dots (core diameter 5 nm) and negatively charged polystyrene nanoparticles (PNP, 20 nm), but ~2‐fold slower than that of positively charged PNP (20 nm). These data indicate that positively or negatively charged ND do not affect barrier function of alveolar epithelium and suggest that ND surface charge affects accumulation in but not translocation across alveolar epithelium. Mechanisms underlying ND entry into/egress from alveolar epithelial cells remain to be determined.(Funded by NIH and Hastings & Whittier Fdns.)

Polystyrene nanoparticle trafficking across MDCK-II

Polystyrene nanoparticles (PNP) cross rat alveolar epithelial cell monolayers via non-endocytic transcellular pathways. To evaluate epithelial cell type-specificity of PNP trafficking, we studied PNP flux across Madin Darby canine kidney cell II monolayers (MDCK-II). The effects of calcium chelation (EGTA), energy depletion (sodium azide (NaN 3) or decreased temperature), and endocytosis inhibitors methyl-β-cyclodextrin (MBC), monodansylcadaverine and dynasore were determined. Amidine-modified PNP cross MDCK-II 500 times faster than carboxylate-modified PNP. PNP flux did not increase in the presence of EGTA. PNP flux at 4°C and after treatment with NaN 3 decreased 75% and 80%, respectively. MBC exposure did not decrease PNP flux, whereas dansylcadaverine- or dynasore-treated MDCK-II exhibited ∼80% decreases in PNP flux. Confocal laser scanning microscopy revealed intracellular colocalization of PNP with clathrin heavy chain. These data indicate that PNP translocation across MDCK-II (1) occurs via clathrin-mediated endocytosis and (2) is dependent on PNP physicochemical properties. We conclude that uptake/trafficking of nanoparticles (NPs) into/across epithelia depends both on properties of the NPs and on the specific epithelial cell type. From the Clinical Editor This basic science study investigates the membrane translocation of polystyrene nanoparticles (PNP) via an epithelial cell monolayer utilizing canine kidney cells. The authors conclude that translocation occurs via clathrin-mediated endocytosis and is dependent on the physicochemical properties of the particular PNP.

Translocation of PEGylated quantum dots across rat alveolar epithelial cell monolayers

BackgroundIn this study, primary rat alveolar epithelial cell monolayers (RAECM) were used to investigate transalveolar epithelial quantum dot trafficking rates and underlying transport mechanisms.MethodsTrafficking rates of quantum dots (PEGylated CdSe/ZnS, core size 5.3 nm, hydrodynamic size 25 nm) in the apical-to-basolateral direction across RAECM were determined. Changes in bioelectric properties (ie, transmonolayer resistance and equivalent active ion transport rate) of RAECM in the presence or absence of quantum dots were measured. Involvement of endocytic pathways in quantum dot trafficking across RAECM was assessed using specific inhibitors (eg, methyl-β-cyclodextrin, chlorpromazine, and dynasore for caveolin-, clathrin-, and dynamin-mediated endocytosis, respectively). The effects of lowering tight junctional resistance on quantum dot trafficking were determined by depleting Ca2+ in apical and basolateral bathing fluids of RAECM using 2 mM EGTA. Effects of temperature on quantum dot trafficking were studied by lowering temperature from 37°C to 4°C.ResultsApical exposure of RAECM to quantum dots did not elicit changes in transmonolayer resistance or ion transport rate for up to 24 hours; quantum dot trafficking rates were not surface charge-dependent; methyl-β-cyclodextrin, chlorpromazine, and dynasore did not decrease quantum dot trafficking rates; lowering of temperature decreased transmonolayer resistance by approximately 90% with a concomitant increase in quantum dot trafficking by about 80%; and 24 hours of treatment of RAECM with EGTA decreased transmonolayer resistance by about 95%, with increased quantum dot trafficking of up to approximately 130%.ConclusionThese data indicate that quantum dots do not injure RAECM and that quantum dot trafficking does not appear to take place via endocytic pathways involving caveolin, clathrin, or dynamin. We conclude that quantum dot translocation across RAECM takes place via both transcellular and paracellular pathways and, based on comparison with our prior studies, interactions of nanoparticles with RAECM are strongly dependent on nanoparticle composition and surface properties.

Alveolar Epithelial Cell Injury Due to Zinc Oxide Nanoparticle Exposure

Although inhalation of zinc oxide (ZnO) nanoparticles (NPs) is known to cause systemic disease (i.e., metal fume fever), little is known about mechanisms underlying injury to alveolar epithelium. Investigate ZnO NP-induced injury to alveolar epithelium by exposing primary cultured rat alveolar epithelial cell monolayers (RAECMs) to ZnO NPs. RAECMs were exposed apically to ZnO NPs or, in some experiments, to culture fluid containing ZnCl₂ or free Zn released from ZnO NPs. Transepithelial electrical resistance (R(T)) and equivalent short-circuit current (I(EQ)) were assessed as functions of concentration and time. Morphologic changes, lactate dehydrogenase release, cell membrane integrity, intracellular reactive oxygen species (ROS), and mitochondrial activity were measured. Apical exposure to 176 μg/ml ZnO NPs decreased R(T) and I(EQ) of RAECMs by 100% over 24 hours, whereas exposure to 11 μg/ml ZnO NPs had little effect. Changes in R(T) and I(EQ) caused by 176 μg/ml ZnO NPs were irreversible. ZnO NP effects on R(T) yielded half-maximal concentrations of approximately 20 μg/ml. Apical exposure for 24 hours to 176 μg/ml ZnO NPs induced decreases in mitochondrial activity and increases in lactate dehydrogenase release, permeability to fluorescein sulfonic acid, increased intracellular ROS, and translocation of ZnO NPs from apical to basolateral fluid (most likely across injured cells and/or damaged paracellular pathways). ZnO NPs cause severe injury to RAECMs in a dose- and time-dependent manner, mediated, at least in part, by free Zn released from ZnO NPs, mitochondrial dysfunction, and increased intracellular ROS.

MAPK activation modulates permeability of isolated rat alveolar epithelial cell monolayers following cyclic stretch.

We cultured (5 days) rat alveolar epithelial cells to investigate the role of mitogen-activated protein kinase (MAPk) signaling in ventilator induced epithelial barrier dysfunction. Cells were stretched to a magnitude of 12% or 37% change in surface area at a rate of 0.25 Hz with and without pretreatment with either the JNK inhibitor SP600125 or the ERK inhibitor U0126. Following stretch (0, 10, 30, or 60 min), MAPk phosphorylation was examined, monolayer permeability to the uncharged tracer carboxyfluorescein measured (0, 10, 60 min of stretch), and occludin expression determined (0 and 60 min of stretch). Stretch to 12%, previously shown not to increase monolayer permeability, did not alter phosphorylation of any MAPk or occludin expression at any time point. Following stretch to 37%, phosphorylation of JNK, ERK, and p38 was significantly higher by 10 minutes than in unstretched monolayers. Phosphorylation of JNK and p38 subsided as stretch continued, and by 30 minutes returned to unstretched levels. Phosphorylation of ERK remained significantly elevated compared to unstretched levels at all stretch durations. Epithelial permeability increased significantly by 10 minutes of stretch compared to unstretched controls, with further significant increases by 60 minutes. Inhibition with U0126 and SP600125 prevented stretch-induced phosphorylation increases of ERK and JNK, respectively, however neither prevented increases in permeability following 10 minutes. Separately, inhibition of JNK or ERK prevented subsequent additional permeability increases as stretch continued to 60 minute time points. Inhibition of JNK, not ERK, prevented loss of occludin, and minimized loss of cell-cell contact following 60 minutes of stretch. These data suggest that stretch-induced JNK signaling modulates epithelial permeability through regulation tight junction protein expression, and is a potential target for clinical treatments during mechanical ventilation.

Mechanisms of Zinc Oxide Nanoparticle (ZONP)‐Induced Alveolar Epithelial Cell (AEC) Injury

We investigated ZONP (20 nm diameter) induction of AEC injury utilizing primary rat alveolar epithelial cell monolayers (RAECM). We assessed injury by measuring transmonolayer resistance (RT) and equivalent short‐circuit current (IEQ), lactate dehydrogenase (LDH) release, intracellular reactive oxygen species (ROS) and mitochondrial activity. We also investigated effects of free Zn2+ released from ZONP on RT and IEQ of RAECM. Results showed that apical exposure to 176 μg/mL ZONP decreased RT and IEQ of RAECM by 100% over 24h, whereas exposure to 11 μg/mL ZONP had little effect. Apical exposure of RAECM for 24h to 176 μg/mL ZONP caused irreversible injury. ZONP effects on RT yielded a half‐maximal concentration of 17 μg/mL. About 8 μg/mL of Zn2+ was found to be released from 176 μg/mL ZONP suspension over 24h. When an equivalent concentration of Zn2+ (as ZnCl2) or supernatant from 24h ZONP suspensions was used to apically expose RAECM, RT was decreased by ~45% after 24h. Apical exposure for 24h to 176 μg/mL ZONP increased LDH release, disrupted cell plasma membrane integrity, decreased mitochondrial activity and increased intracellular ROS production. These data indicate that apical ZONP exposure of RAECM leads to severe cellular injury involving increased oxidative stress and disruption of cell membrane integrity, effects due in part to free Zn2+ released from ZONP. (Funded by: NIH; Hastings & Whittier Foundations.)

Mechanisms of Alveolar Epithelial Translocation of a Defined Population of Nanoparticles

To explore mechanisms of nanoparticle interactions with and trafficking across lung alveolar epithelium, we utilized primary rat alveolar epithelial cell monolayers (RAECMs) and an artificial lipid bilayer on filter model (ALBF). Trafficking rates of fluorescently labeled polystyrene nanoparticles (PNPs; 20 and 100 nm, carboxylate (negatively charged) or amidine (positively charged)-modified) in the apical-to-basolateral direction under various experimental conditions were measured. Using confocal laser scanning microscopy, we investigated PNP colocalization with early endosome antigen-1, caveolin-1, clathrin heavy chain, cholera toxin B, and wheat germ agglutinin. Leakage of 5-carboxyfluorescein diacetate from RAECMs, and trafficking of (22)Na and (14)C-mannitol across ALBF, were measured in the presence and absence of PNPs. Results showed that trafficking of positively charged PNPs was 20-40 times that of negatively charged PNPs across both RAECMs and ALBF, whereas translocation of PNPs across RAECMs was 2-3 times faster than that across ALBF. Trafficking rates of PNPs across RAECMs did not change in the presence of EGTA (which decreased transepithelial electrical resistance to zero) or inhibitors of endocytosis. Confocal laser scanning microscopy revealed no intracellular colocalization of PNPs with early endosome antigen-1, caveolin-1, clathrin heavy chain, cholera toxin B, or wheat germ agglutinin. Leakage of 5-carboxyfluorescein diacetate from alveolar epithelial cells, and sodium ion and mannitol flux across ALBF, were not different in the presence or absence of PNPs. These data indicate that PNPs translocate primarily transcellularly across RAECMs, but not via known major endocytic pathways, and suggest that such translocation may take place by diffusion of PNPs through the lipid bilayer of cell plasma membranes.

Trafficking of Silica Nanoparticles Across the Alveolar Epithelial Barrier

Polystyrene nanoparticles (PNP) translocate across primary cultured rat alveolar epithelial cell monolayers (RAECM) in a surface charge‐ and size‐dependent manner. In this study, we investigated interactions of fluorescently labeled silica nanoparticles (SNP, 42 and 105 nm) with RAECM. Effects of apical 176 µg/mL SNP on transmonolayer resistance (Rt) and equivalent short‐circuit current (Ieq) were measured as functions of time. SNP fluxes (apical‐to‐basolateral) were determined in the presence and absence of the caveolin‐ or clathrin‐mediated endocytosis inhibitors methyl‐β ‐cyclodextrin (MβC) or chlorpromazine (CHL), respectively. Results showed that: i) SNP did not elicit changes in Rt and Ieq for up to 24 hr, ii) 42 nm SNP flux >105 nm SNP flux, iii) fluxes of negatively charged 20 and 100 nm PNP were 60‐80 fold less than fluxes of 42 and 105 nm SNP, iv) neither MβC nor CHL affected SNP fluxes, and v) confocal microscopy after exposure to SNP for 24 hr revealed intracellular localization of SNP. These data indicate that: (1) SNP do not injure RAECM, (2) translocation of SNP is transcellular and size‐dependent, (3) SNP translocation is much faster than comparably‐sized PNP translocation, and (4) SNP trafficking does not appear to take place via endocytosis pathways requiring clathrin or caveolin. We conclude that nanoparticle interactions with alveolar epithelium are at least in part composition‐dependent.Funded by: NIH & Hastings Foundation

Polystyrene nanoparticle trafficking across alveolar epithelium

We investigated trafficking of polystyrene nanoparticles (PNP; 20 and 100 nm; carboxylate, sulfate, or aldehyde-sulfate modified [negatively charged] and amidine-modified [positively charged]) across rat alveolar epithelial cell monolayers (RAECM). Apical-to-basolateral fluxes of nanoparticles were estimated as functions of apical PNP concentration ([PNP]) and temperature. Uptake of nanoparticles into RAECM was determined using confocal microscopy. Fluxes increased as charge density became less negative/more positive, with positively charged PNPs trafficking 20–40 times faster than highly negatively charged PNP of comparable size. Trafficking rates decreased with increasing PNP diameter. PNP fluxes tended to level off at high apical [PNP]. Fluxes at 4°C were significantly lower than those at 37°C. Confocal microscopy revealed nanoparticles localized to cell cytoplasm, whereas cell junctions and nuclei appeared free of PNP. These data indicate that (1) trafficking of PNP across RAECM is strongly influenced by charge density, size, and temperature, (2) PNP translocate primarily transcellularly, and (3) PNP translocation requires cellular energy.

Dilution potential (DP) across rat alveolar epithelial cell monolayers

In this study, we estimated permeability ratio (PNa/PCl) between Na and Cl for paracellular pathways of primary rat alveolar epithelial cell monolayers (RAECM). PNa/PCl was estimated using Goldman‐Hodgkin‐Katz equation and DP measured across RAECM at 5–7 days. To inhibit Na pump activities, RAECM were bathed on both sides with K‐free Ringer's solution (KFRS, in which 5 mM KCl is replaced by equimolar NaCl). DP was measured after lowering apical [NaCl] tenfold by replacing apical fluid with a 9:1 mixture of isotonic K‐free mannitol solution and KFRS. Osmolarity of all solutions was adjusted to 300 mOsm with mannitol. DP was further corrected for liquid junction potential of voltage‐sensing electrodes across blank filters. Results show that equivalent short‐circuit current across RAECM bathed on both sides with KFRS is not significantly different from zero with electrical resistance >1,000 Ωcm2. When apical [NaCl] was lowered tenfold, corrected DP ∼ −13 mV (apical as reference) was observed, yielding PNa/PCl ∼1.8. For comparison, the free solution diffusivity ratio of Na to Cl is ∼0.7, while paracellular PNa/PCl of various epithelial barriers ranges from ∼0.2 and ∼0.5 for frog skin and bullfrog lung to ∼1.3 and ∼10 for rabbit proximal tubule and rat jejunum, respectively. These data indicate that passive ion conductance through tight junctions of RAECM is greater for Na than Cl. (Support: NIH and Hastings Foundation.)

Characterization of protein factor(s) in rat bronchoalveolar lavage fluid that enhance insulin transport via transcytosis across primary rat alveolar epithelial cell monolayers

The aim of this study was to characterize factor(s) in rat bronchoalveolar lavage fluid (BALF) that enhance(s) insulin transport across primary rat alveolar epithelial cell monolayers (RAECM) in primary culture. BALF was concentrated 7.5-fold using the Centricon device and the retentate was used to characterize the factor(s) involved in enhancing apical-to-basolateral transport of intact 125I-insulin across various epithelial cell monolayers. These factor(s) enhanced transport of intact insulin across type II cell-like RAECM (3-fold increase) and type I cell-like RAECM (2-fold increase), but not across Caco-2 or MDCK cell monolayers. The insulin transport-enhancing factor(s) were temperature- and trypsin-sensitive. The mechanism of enhancement did not seem to involve paracellular transport or fluid-phase endocytosis, since fluxes of sodium fluorescein and FITC-dextran (70 kDa) were not affected by the factor(s) in the apical bathing fluid. BALF enhancement of intact 125I-insulin transport was abolished at 4 °C and in the presence of monensin, suggesting involvement of transcellular pathways. Sephacryl S-200 purification of BALF retentate, followed by LC-MS/MS, indicated that the high molecular weight (>100 kDa) fractions (which show some homology to alpha-1-inhibitor III, murinoglobulin gamma 2, and pregnancy-zone protein) appear to facilitate transcellular transport of insulin across RAECM.