Neurite transection produces cytosolic oxidation, which enhances plasmalemmal repair.

To survive, cells must rapidly repair (seal) plasmalemmal damage. Cytosolic oxidation has been shown to increase cell survival in some cases and produce cell death in other protocols. An antioxidant (melatonin; Mel) has been reported to decrease the probability of sealing plasmalemmal damage. Here we report that plasmalemmal damage produces cytosolic oxidation, as assayed by methylene blue (MB) color change in rat B104 hippocampal cells. Plasmalemmal sealing is affected by duration of Ca²âº deprivation and length of exposure to, and concentration of, oxidizing agents such as H2O2 and thimerosal (TH). Cytosolic oxidation by 10 µM to 50 mM H2O2 or 100 µM to 2 mM TH increases the probability of Ca²âº-dependent plasmalemmal sealing, whereas higher concentrations of H2O2 decrease sealing probability and also damage uninjured cells. We also show that antioxidants (Mel, MB) or reducing agents (dithiothreitol) decrease sealing. Proteins, such as protein kinase A, SNAP-25, synaptobrevin, and N-ethylmaleimide-sensitive factor (previously reported to enhance sealing in other pathways), also enhance sealing in this oxidation pathway. In brief, our data show that plasmalemmal damage produces cytosolic oxidation that increases the probability of plasmalemmal sealing, which is strongly correlated with cell survival in other studies. Our results may provide new insights into the etiology and treatment of oxidation-dependent neurodegenerative disorders, such as Parkinson's, Huntington's, and Alzheimer's diseases.

Cellular mechanisms of plasmalemmal sealing and axonal repair by polyethylene glycol and methylene blue.

Mammalian neurons and all other eukaryotic cells endogenously repair traumatic injury within minutes by a Ca²âº-induced accumulation of vesicles that interact and fuse with each other and the plasmalemma to seal any openings. We have used uptake or exclusion of extracellular fluorescent dye to measure the ability of rat hippocampal B104 cells or rat sciatic nerves to repair (seal) transected neurites in vitro or transected axons ex vivo. We report that endogenous sealing in both preparations is enhanced by Ca²âº-containing solutions and is decreased by Ca²âº-free solutions containing antioxidants such as dithiothreitol (DTT), melatonin (MEL), methylene blue (MB), and various toxins that decrease vesicular interactions. In contrast, the fusogen polyethylene glycol (PEG) at 10-50 mM artificially seals the cut ends of B104 cells and rat sciatic axons within seconds and is not affected by Ca²âº or any of the substances that affect endogenous sealing. At higher concentrations, PEG decreases sealing of transected axons and disrupts the plasmalemma of intact cells. These PEG-sealing data are consistent with the hypothesis that lower concentrations of PEG directly seal a damaged plasmalemma. We have considered these and other data to devise a protocol using a well-specified series of solutions that vary in tonicity, Ca²âº, MB, and PEG content. These protocols rapidly and consistently repair (PEG-fuse) rat sciatic axons in completely cut sciatic nerves in vivo rapidly and dramatically to restore long-lasting morphological continuity, action potential conduction, and behavioral functions.

Ascertainment of pesticide exposures of migrant and seasonal farmworker children: findings from focus groups.

BACKGROUND: To design questionnaires for epidemiologic research among children of migrant farmworkers, researchers need to consider ways to best solicit information about pesticide exposures. METHODS: Bilingual facilitators conducted five focus groups with either migrant farmworker mothers or their children (age range 8-16 years) in southern Texas and northeastern Colorado. Guided questions were used to assess activities of migrant farmworker children and the ways to best elicit information about exposure to pesticides. RESULTS: Participants reported a large number of activities that may potentially expose children to pesticides through both direct and indirect routes. Prompting, indirect questions about chemical use, and use of local and trusted facilitators increased information elicited from focus group participants. CONCLUSIONS: These focus groups helped to provide information for developing questionnaire items related to pesticide exposure among migrant farmworker children, and highlighted the importance of using bilingual community interviewers and including children as respondents.

Prenatal exposure to pesticides: a feasibility study among migrant and seasonal farmworkers.

BACKGROUND: Migrant and seasonal farmworkers have a high potential for pesticide exposures, yet are rarely included in epidemiologic studies. This study examined the feasibility of assessing prenatal exposures to pesticides and other compounds in pregnant Hispanic farmworkers. METHODS: Nine women completed a survey about work experiences during pregnancy. Maternal urine, cord blood, and placenta samples were obtained at delivery for analysis of 51 analytes, including 6 phenoxy acid or triazine herbicides, 21 organochlorine insecticides, 10 PCBs, and 14 volatile organic compounds. RESULTS: Seven of 51 analytes were found in the biological samples. DDE, DDT, dichlorbenzene, toluene, trimethylbenzene, and endosulfan sulfate were detected in cord blood samples, and 2,4-D in urine from one or more women. CONCLUSIONS: We documented the feasibility of following farmworkers to assess in utero exposure to pesticides and other contaminants, and demonstrated exposure to these compounds. Difficulties in measuring pesticides with short half lives were noted.

Tracing migrant farmworkers in Starr County, Texas.

BACKGROUND: In response to The National Cancer Institute (NCI) concerns about the ability to conduct studies among migrant farmworkers, this study evaluated the feasibility of identifying migrant farmworkers in their home state and tracing them over an extended period of time. METHODS: In 1995, a group of 196 persons who had classified themselves as "migrant farmworkers" in two earlier chronic disease studies was identified. The primary objective of the current study was to determine the proportion of these farmworkers who could be located in 1995-1996. RESULTS: Of these farmworkers, 163 were located and were living (83.2%), 15 had died (7.6%), and 18 (9.2%) were lost to follow-up. CONCLUSIONS: The excellent follow-up rate was due in part to the high participation rates among persons contacted for information, stability of the farmworkers' permanent homes, predictable timing of migration, and a longstanding health research program with established community contacts.

Adolescent occupational injuries: Texas, 1990-1996.

BACKGROUND: A comprehensive surveillance system for occupational injuries to adolescents does not exist in Texas, as in most states. Therefore, the magnitude, severity, nature, and source of injuries to working adolescents have not been well described in Texas. METHODS: The investigators used three data sources to investigate work-related injuries and deaths in Texas: (1) Texas Workers' Compensation Commission (TWCC) claims data from 1991 through April 1996; (2) 1993 TWCC/Bureau of Labor Statistics (BLS) Annual Survey of Occupational Injuries and Illnesses; and (3) work-related fatalities identified from Texas death certificates from 1990-1995. RESULTS: There were 9,027 injuries reported to the TWCC for adolescents 14-17 years of age during slightly more than 5 years. Injuries for which indemnity payments were made (more than 7 days out of work) occurred among 21.7% of the adolescents. Based on BLS data in 1993, of 992 non-fatal injuries involving days away from work, 35% were caused by contact with objects, 27% by bodily reaction, and 24% by falls. Two-thirds of these injuries occurred while working in eating and drinking places and grocery stores. Three-quarters of the 30 deaths from 1990-1995 were accounted for equally by motor vehicle and homicide. CONCLUSIONS: In conclusion, a substantial number of adolescents are injured or killed in the workplace each year in Texas. Although improved population-based surveillance is needed, sufficient knowledge exists to begin prevention efforts now.

Ca(2+)-dependent p47phox translocation in hydroperoxide modulation of the alveolar macrophage respiratory burst.

Oxidative stress produces dual effects on the respiratory burst of rat alveolar macrophages. Preincubation with hydroperoxide concentrations [H2O2 or tert-butyl hydroperoxide (t-BOOH); < 50 microM] enhances stimulation of the respiratory burst, whereas higher concentrations inhibit stimulation. Both the enhancement and inhibition are markedly attenuated by buffering t-BOOH-induced changes in intracellular Ca2+ concentration ([Ca2+]i). Phosphorylation of the NADPH oxidase component p47phox and its translocation from cytoplasm to plasma membrane are essential in respiratory burst activation. Phorbol 12-myristate 13-acetate (PMA)-stimulated p47phox phosphorylation was negligibly affected by 25 or 100 microM t-BOOH. Nonetheless, 25 microM t-BOOH increased PMA-stimulated p47phox translocation, whereas 100 microM t-BOOH decreased PMA-stimulated translocation. In unstimulated cells, however, neither phosphorylation nor translocation of p47phox was affected by t-BOOH. Buffering of the t-BOOH-mediated changes of [Ca2+]i abolished the effects of t-BOOH on PMA-stimulated translocation in parallel to effects upon the respiratory burst. The results suggest that the dual effects of hydroperoxides are mediated, in part, by Ca(2+)-dependent processes affecting the assembly of the respiratory burst oxidase at steps that are separate from p47phox phosphorylation.

Generation of glycolaldehyde from guinea pig airway epithelial monolayers exposed to nitrogen dioxide and its effects on sodium pump activity.

Pulmonary injury from nitrogen dioxide (NO2) may in part be related to the generation of aldehydic compounds, which bind with cellular proteins and subsequently impair or inhibit cell function. We examined the generation of aldehydes from guinea pig tracheobronchial epithelial (GPTE) cell monolayers exposed to NO2. With the use of dinitrophenylhydrazine (DNP) to derivatize aldehydic compounds, glycolaldehyde, a two carbon alpha-hydroxyaldehyde, was identified in elevated levels in the basolateral fluid from monolayers exposed to NO2. DNP-glycolaldehyde levels were 81.2 +/- 2.7 and 234.0 +/- 42.6 nM in response to a 1-hr exposure to 1 and 5 ppm NO2, respectively, as compared to an air-control value of 20.3 +/- 6.8 nM. Taking into account dilution and reactivity, cellular glycolaldehyde levels could have reached as high as 3 mM for the 60-min exposure period (i.e., 0.05 mM/min). The effects of exogenous glycolaldehyde on GPTE ouabain-sensitive basolateral 86Rb uptake (an index of Na+,K(+)-ATPase activity) were examined and compared with the actions of NO2 exposure. Bolus addition of glycolaldehyde to the basolateral fluid at concentrations > or = 5 mM led to an inhibition of ouabain-sensitive 86Rb uptake, while lower concentrations had no effect. the effects of exogenous glycolaldehyde differ from NO2 exposure, which led to a sustained elevation of ouabain-sensitive 86Rb uptake with presumed generation of glycolaldehyde at a continuous low level. Glycolaldehyde does not appear to play a significant role in the acute alterations of sodium pump activity, suggesting that the NO2-induced changes in Na+,K(+)-ATPase activity of GPTE monolayers probably are further mediated by other lipid peroxidation products/oxidation processes yet to be identified.

Differential enhancement of gamma-glutamyl transpeptidase and gamma-glutamylcysteine synthetase by tert-butylhydroquinone in rat lung epithelial L2 cells.

Sublethal quinone-mediated oxidative stress stimulates increases in the activities and mRNA levels of gamma-glutamyl transpeptidase (GGT) and gamma-glutamylcysteine synthetase (GCS) in rat lung epithelial L2 cells [Kugelman, A. et al. 1994. Am. J. Respir. Cell Mol. Biol. 11:586-592; Shi, M. M. et al. 1994. J. Biol. Chem. 269:26512-26517]. The present study demonstrated that the quinone-induced increases in these two enzymes were differentially regulated. L2 cells were exposed to various concentrations of tertiary-butylhydroquinone (TBHQ) for different periods of times. TBHQ increased the activities and the mRNAs for GGT and the catalytic subunit of GCS; however, the time- and concentration-dependencies differed. With 50 microM TBHQ, GCS activity increased significantly by 6 h whereas the activity of GGT was not increased until later. Under the same conditions, the highest GCS-mRNA level observed was at 6 h whereas the mRNA level of GGT increased after 6 h, reached a higher level at 12 h, and then returned to the control level by 24 h. Differences were also observed in the concentration-dependence of mRNA increases between the GGT and GCS. Actinomycin D (an inhibitor of RNA synthesis) abolished the increase of GCS-mRNA but not the increase in GGT-mRNA, suggesting a difference in regulation by TBHQ between these two genes. Nuclear run-on experiments confirmed that the increase of GCS-mRNA, but not GGT-mRNA was due to increased transcription. The increase in GGT-mRNA probably results from a decreased degradation rate. The differences between these two enzymes demonstrate how cells can use multiple mechanisms for regulating gene expression in response to oxidative stress.

Increased gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase activities enhance resistance of rat lung epithelial L2 cells to quinone toxicity.

Tert-butylhydroquinone (TBHQ) is a monofunctional Phase II enzyme inducer, which produces reactive oxygen species. Incubation with a sublethal concentration of TBHQ increased the activities of both gamma-glutamyl transpeptidase (GGT) and gamma-glutamylcysteine synthetase (GCS), although the mechanisms are different (Liu and colleagues, accompanying manuscript). In this study, we found that TBHQ increased intracellular glutathione (GSH) content in rat lung epithelial L2 cells. L2 cells pretreated with a nontoxic concentration of TBHQ (50 microM) acquired resistance to a subsequent challenge with a normally lethal concentration of TBHQ (200 microM). Pretreatment with L-buthionine S,R-sulfoximine (BSO), an inhibitor of GCS, prevented the TBHQ-induced increase in GSH and markedly diminished resistance to 200 microM TBHQ. Similarly, pretreatment with acivicin, an inhibitor of GGT, also prevented the TBHQ-induced increase in GSH and markedly diminished resistance to 200 microM TBHQ. Nevertheless, blockage of GGT by acivicin could be bypassed using 2-oxothiazolidine-4-carboxylate (procysteine) to provide the cell with a source of cysteine. This allowed an increase in GSH and restored resistance in the TBHQ-pretreated cells. The results suggest that elevation of GCS and GGT activities participated in acquired resistance to quinone toxicity.

Enhancement of airway epithelial Na+,K(+)-ATPase activity by NO2 and protective role of nordihydroguaiaretic acid.

We examined the effects of nitrogen dioxide (NO2) on guinea pig tracheobronchial (GPTE) ouabain-sensitive 86Rb uptake, as an index of Na+,K(+)-ATPase activity, and specific [3H]ouabain binding. A 1-h exposure of GPTE monolayers to 5 ppm NO2 increased ouabain-sensitive 86Rb uptake (nmol.mg protein-1.30 min-1) to 512 +/- 39 compared with an air-control value of 278 +/- 20. Similarly, 1 ppm NO2 increased 86Rb uptake to 336 +/- 19 from an air control of 219 +/- 31. The specific [3H]ouabain-binding capacity (Bmax) for monolayers exposed to 5 ppm NO2 was increased to 23.2 +/- 1.2 pmol/mg protein in comparison with an air-control value of 18.4 +/- 0.4; however, there was no change at 1 ppm NO2. Binding constants (Kd) for 1 or 5 ppm NO2 were increased to 0.64 +/- 0.02 and 0.79 +/- 0.08 microM, respectively, in comparison with an air-control value of 0.53 +/- 0.02 microM. Changes of Bmax and Kd may be consistent with a recruitment of latent pumps to the basolateral cell plasma membrane and/or increased turnover of the sodium pump. However, the increase of Bmax was no more than 126% of the air control, while 86Rb uptake increased to 184%, suggesting that an increased turnover is the more predominant effect. Incubation of GPTE monolayers during NO2 exposure with nordihydroguaiaretic acid, an antioxidant, blocked the increase of ouabain-sensitive 86Rb uptake almost completely and partially protected transepithetial resistance, suggesting that lipid peroxidation processes may play a role in alterations of airway epithelial barrier and active ion transport properties.

Collaborative QI in community-based long term care.

BACKGROUND: In a statewide demonstration project funded by The Robert Wood Johnson Foundation, the state of Indiana, vendors, clients and Indiana University researchers began working together in 1992 to use quality improvement (QI) techniques to improve the delivery of community-based long term care services. QI STRATEGIES: These collaborators, working with state Area Agencies on Aging (AAAs) case managers, are implementing two strategies--Normative Treatment Planning (NTP), which standardizes the clinical assessment of client needs and the prescription of services by case managers, and the Client Feedback System (CFS), a systematic method for obtaining feedback from long term care clients on the quality of in-home services. CURRENT STATUS: This community-based long term care project has been implemented in AAAs throughout the state of Indiana. In January 1995 the state's 16 AAAs were randomly assigned to four experimental or control groups to assess the project's effectiveness. In the interim, clients are surveyed by telephone every six months to evaluate their satisfaction with services and clinical needs. LESSONS LEARNED: The experience suggests several lessons: (1) build on existing and successful activities; (2) involve a wide range of participants, not just innovators; (3) obtain buy-in from trade and professional associations that represent program participants; (4) turn national attention given to the program into an asset; (5) conduct separate data collection to evaluate an intervention's success; (6) visit the field often; (7) pay as much attention to program implementation as to development; and (8) provide ongoing, informal educational opportunities for the field. SUMMARY AND CONCLUSIONS: This project has resulted in significant movement toward a shared quality improvement vocabulary, information system, and a shared vision of high-quality home care.

Development and characterization of rabbit tracheal epithelial cell monolayer models for drug transport studies.

PURPOSE: The objective of this study was to investigate how culture conditions would affect the morphological, functional, and permeability characteristics of rabbit tracheal epithelial cell layers being considered for drug transport studies. METHODS: Rabbit tracheocytes were isolated by protease treatment and plated onto collagen-treated permeable supports at 1.3 x 10(6) cells/cm2. After 24 hr, cell layers were cultured either air-interfaced (AIC) on their apical surface or under conventional liquid covered conditions (LCC). RESULTS: Scanning electron microscopy revealed mature cilia in AIC cell layers and ciliated cells denuded of cilia in LCC cell layers. Compared with LCC, AIC cell layers (n = 20) achieved a significantly higher peak equivalent short-circuit current (74.1 +/- 6.5 vs. 51.6 +/- 3.4 microA/cm2), a higher potential difference (70.9 +/- 2.8 vs. 60.5 +/- 3.0 mV), and a lower peak transepithelial electrical resistance (1.1 +/- 0.03 vs, 1.5 +/- 0.02 kohms.cm2). About 70% of the short-circuit current in AIC was amiloride-sensitive whereas < 10% was furosemide-sensitive, similar to that found in native tissue. The corresponding values in LCC were 50% and 46%. The permeability of both AIC and LCC to lipophilic solutes (dexamethasone and propranolol) was similar, whereas the permeability of hydrophilic solutes (mannitol, sucrose, and albuterol) in AIC was only half that in LCC. CONCLUSIONS: Given the striking similarity in morphological and functional characteristics of the AIC to those in the in vivo situation, the AIC is favored as an in vitro model for future drug transport studies.

Release of aldehydes from rat alveolar macrophages exposed in vitro to low concentrations of nitrogen dioxide.

This study demonstrated that aldehydes are released into the extracellular medium when alveolar macrophages (AM) are exposed to nitrogen dioxide (NO2) at concentrations that impair cell function but do not cause cell death. Butanal, glycolaldehyde, 4-hydroxynonenal, pentanal, pentenal, and hexanal were found. Dinitrophenylhydrazine (DNP) derivitization, thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectrometry were used to identify the products. Some of the aldehydes have potential toxicity and may be responsible, in part, for altered AM function observed following NO2 exposure.

Modulation of ADP-stimulated inositol phosphate metabolism in rat alveolar macrophages by oxidative stress.

The present study examined alterations of adenosine-5-diphosphate (ADP)-stimulated inositol phosphate (IP3) metabolism and the respiratory burst of rat alveolar macrophages (AM) under oxidant stress using the model oxidant, tert-butyl hydroperoxide (tBOOH). Isolated AM were maintained in a system with an air-liquid interface, which approximates the lung environment. tBOOH produced a dual effect on the ADP-stimulated respiratory burst of these AM. Pretreatment of these AM with 50 microM tBOOH for 15 min led to a significant enhancement of the respiratory burst while significant inhibition was observed with 200 microM tBOOH. Treatment of AM with 100 microM ADP led to a significant increase in the generation of IP3, reaching a maximum at 30 s. In contrast, treatment of AM with 50 or 200 microM tBOOH did not stimulate IP3 generation during a 15-min period. Pretreatment of AM with 50 microM tBOOH for 15 min had no effect on ADP-stimulated IP3 generation. Preincubation with 200 microM tBOOH significantly inhibited generation of IP3 in response to ADP stimulation; however, this probably did not contribute to inhibition of the respiratory burst. The results suggest that production of IP3 may participate in stimulation of the respiratory burst by ADP but that enhancement of the respiratory burst by 50 microM tBOOH probably did not involve alteration of IP3 production.

Detecting and identifying volatile aldehydes as dinitrophenylhydrazones using gas chromatography mass spectrometry.

The detection of aldehydes has become an important measure of lipid oxidation in biological milieu. Aldehyde 2,4-dinitrophenylhydrazones are easily prepared and readily purified by HPLC and/or TLC and have proven useful for the detection of aldehydes. The lower limit of detection for dinitrophenylhydrazones was significantly reduced by using gas chromatography-mass spectrometric (GC-MS) techniques. Individual dinitrophenylhydrazones were readily separated by GC and detected by both positive and negative ion MS. The two major ions in negative ion spectra were the 182 m/z fragment ion and the molecular ion. Positive ion spectra showed strong ions corresponding to the protonated molecular ion and a protonated iminium ion. The greatest sensitivity was obtained with negative ion detection (10 pg per injection). However, more structural information was obtained from analysis of the positive ion spectra. Dinitrophenylhydrazones of hydroxyaldehydes, like 4-hydroxynonenal, were analyzed after converting the dinitrophenylhydrazones into trimethylsiloxylethers. GC-MS with negative ion detection was used to identify and quantitate the release of 4-hydroxynonenal by alveolar macrophages exposed to nitrogen dioxide.

Dual effect of nitrogen dioxide on barrier properties of guinea pig tracheobronchial epithelial monolayers cultured in an air interface.

Nitrogen dioxide (NO2) is an oxidant gas that may injure the airway epithelial lining, leading to decrements in barrier and active ion transport properties. The present studies examined alterations of bioelectric properties and solute flux by guinea pig tracheobronchial epithelial (GPTE) monolayers exposed in vitro to NO2. Confluent GPTE monolayers were exposed to NO2 levels between 0.5 and 5 ppm, while controls were exposed to air. Following exposure, monolayers were mounted in Ussing chambers for measurement of transepithelial resistance (Rte) and short-circuit current (SCC). A 1-h exposure to 1 ppm NO2 significantly increased SCC to 131.3 +/- 8.7% of air controls, while Rte with a value of 109.3 +/- 13.8% was unchanged. In contrast, a 1-h exposure to 2 or 5 ppm NO2 significantly decreased Rte to 39.0 +/- 1.6 or 35.5 +/- 7.3% of air controls, respectively, while SCC values of 140.3 +/- 10.4 or 153.3 +/- 8.6%, respectively, were also significantly elevated. A 1-h exposure to 2 or 5 ppm NO2 significantly increased sucrose permeability across GPTE monolayers to 446.8 +/- 117 or 313.3 +/- 39.5% of air controls, respectively, while glycerol permeability was unchanged. In contrast, a 1-h exposure to 1 ppm NO2 produced no alterations of sucrose or glycerol flux. The SCC of control GPTE monolayers (1-h air exposure) consisted of 50% bumetanide-sensitive and 40% amiloride-sensitive current; exposure for 1 h to 2 ppm NO2 led to no changes in the corresponding SCC components. Active ion transport (i.e., SCC) across the airway epithelium was significantly increased after exposure to NO2 levels < or = 1 ppm with no change of paracellular pathways for diffusion, suggesting that this reactive gas alters cell membrane function. The increased SCC may lead to impairment of fluid balance and mucociliary clearance. NO2-mediated tissue injury with levels > or = 2 ppm primarily affects passive airway epithelial barrier functions, probably by altering tight junctions, which could result in increased transepithelial solute and fluid leakage in vivo.

Air-interface cultures of guinea pig airway epithelial cells: effects of active sodium and chloride transport inhibitors on bioelectric properties.

To systematically study airway epithelial barrier properties and active ion transport processes during challenge to environmental toxicants, in vitro models that closely resemble airway epithelium in vivo are required. Guinea pig tracheobronchial epithelial (GPTE) cells cultured in an air interface form a tight, confluent monolayer, which may be a more suitable model for the studies of the airway epithelial barrier in vivo. In the present study, bioelectric properties of such GPTE cell monolayers were characterized with the use of pharmacological agents. Treatment of the basolateral side with ouabain completely abolished the short-circuit current (SCC), while apical addition had little effect. Apical addition of amiloride abolished 75% of the SCC, while basal addition had little effect. Treatment of the basolateral side with furosemide or bumetanide reduced the SCC by 20%, while apical addition had no effect. Apical or basolateral terbutaline increased SCC threefold with identical time courses; results with furosemide suggest a preferential stimulation of Cl- secretion. In contrast to GPTE monolayers, canine tracheal epithelial monolayers, under short-circuit conditions, primarily secrete Cl-, possibly due to apparent species differences. The predominance of Na+ reabsorption suggests that GPTE monolayers cultured in an air interface may share similar transport properties to human airway epithelium.

Formation of tight monolayers of guinea pig airway epithelial cells cultured in an air-interface: bioelectric properties.

In this study, we have developed an air-interface culture system in which guinea pig tracheobronchial epithelial (GPTE) cells rapidly form tight monolayers. Enzymatically isolated GPTE cells were plated on collagen-treated polycarbonate microporous cell culture inserts at a density of 10(6) cells/cm2 (day 0). Bioelectric properties of cultures grown in an air-interface were compared with those covered by medium. On day 1 for air-interface cultures, apical fluid was removed and basolateral fluid was replenished. For cultures covered by medium, varying volumes of apical fluid were used. On days 4 and 5 after plating, confluent GPTE monolayers in either liquid-covered or air-interface cultures exhibited similar monolayer resistance values > or = 1.0 kohm-cm2. However, the equivalent short-circuit current (Ieq) was significantly higher in air-interface cultures than those covered with medium on days 4 and 5. The Ieq in air-interface cultures on day 4 was 12.9 microA/cm2. These confluent GPTE cell monolayers cultured in air-interface could be a useful tool for studies of changes in bioelectric and ion transport properties in response to injury and mediators of inflammation.