Reversible Hypothermia in a Drug-naive Inpatient with Alzheimer's Disease Receiving Pipamperone.

Hypothermia is a potentially life-threatening side effect of antipsychotic drugs, especially those with strong 5-HT2 antagonist properties. However, the exact underlying mechanism is still under debate. We discuss a case of hypothermia following pipamperone treatment in an elderly female inpatient with Alzheimer's disease, which occurred at day 4 after medication onset and vanished after dose reduction. Thus, this case demonstrates 1) the importance of monitoring body temperature even in low-potency antipsychotics, at least in the elderly, and 2) that in some cases, dose reduction may be a sufficient countermeasure.

Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis.

8-Oxoguanine DNA glycosylase (Ogg1) repairs 8-oxo-7,8-dihydroxyguanine (8-oxoG), one of the most abundant DNA adducts caused by oxidative stress. In the mitochondria, Ogg1 is thought to prevent activation of the intrinsic apoptotic pathway in response to oxidative stress by augmenting DNA repair. However, the predominance of the beta-Ogg1 isoform, which lacks 8-oxoG DNA glycosylase activity, suggests that mitochondrial Ogg1 functions in a role independent of DNA repair. We report here that overexpression of mitochondria-targeted human alpha-hOgg1 (mt-hOgg1) in human lung adenocarcinoma cells with some alveolar epithelial cell characteristics (A549 cells) prevents oxidant-induced mitochondrial dysfunction and apoptosis by preserving mitochondrial aconitase. Importantly, mitochondrial alpha-hOgg1 mutants lacking 8-oxoG DNA repair activity were as effective as wild-type mt-hOgg1 in preventing oxidant-induced caspase-9 activation, reductions in mitochondrial aconitase, and apoptosis, suggesting that the protective effects of mt-hOgg1 occur independent of DNA repair. Notably, wild-type and mutant mt-hOgg1 coprecipitate with mitochondrial aconitase. Furthermore, overexpression of mitochondrial aconitase abolishes oxidant-induced apoptosis whereas hOgg1 silencing using shRNA reduces mitochondrial aconitase and augments apoptosis. These findings suggest a novel mechanism that mt-hOgg1 acts as a mitochondrial aconitase chaperone protein to prevent oxidant-mediated mitochondrial dysfunction and apoptosis that might be important in the molecular events underlying oxidant-induced toxicity.

Fibroblast growth factor-10 upregulates Na,K-ATPase via the MAPK pathway.

We studied the effects of fibroblast growth factor (FGF-10) on alveolar epithelial cell (AEC) Na,K-ATPase regulation. Within 30 min FGF-10 increased Na,K-ATPase activity and alpha1 protein abundance by 2.5-fold at the AEC plasma membrane. Pretreatment of AEC with the mitogen-activated protein kinase (MAPK) inhibitor U0126, a Grb2-SOS inhibitor (SH3-b-p peptide), or a Ras inhibitor (farnesyl transferase inhibitor (FTI 277)), as well as N17-AEC that express a Ras dominant negative protein each prevented FGF-10-mediated Na,K-ATPase recruitment to the AEC plasma membrane. Accordingly, we provide first evidence that FGF-10 upregulates (short-term) the Na,K-ATPase activity in AEC via the Grb2-SOS/Ras/MAPK pathway.

Inhibition and stimulation of phospholipid scrambling activity. Consequences for lipid asymmetry, echinocytosis, and microvesiculation of erythrocytes.

An increase of the intracellular Ca(2+) concentration in erythrocytes is known to activate rapid nonspecific bidirectional translocation of membrane-inserted phospholipid probes and to decrease the asymmetric distribution of endogenous membrane phospholipids. These scrambling effects are now shown to be suppressed by pretreatment of cells with the essentially impermeable reagents 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 2,4,6-trinitrobenzenesulfonate. The inhibitory effects are no longer observed during renewed activation of scrambling following a first transient activation by Ca(2+). Assuming the involvement of the human scramblase, this suggests a conformational alteration of this protein during activation by Ca(2+). Marked suppression of scrambling activity is also observed in cells pretreated with the disulfide reducing agent dithioerythritol which can be reverted by the SH oxidizing agent diamide. This indicates the importance of intramolecular and/or intersubunit disulfide bonds for the function of the scramblase. On the other hand, treatment of cells with the SH reagents N-ethylmaleimide and phenylarsine oxide enhances Ca(2+)-activated scrambling and diminution of asymmetry of membrane phospholipids. This suggests an allosteric connection of several protein SH groups to the translocation mechanism. The inhibitors retain their strong suppressive effects. Besides covalent modification, addition of oligomycin highly stimulates and addition of clotrimazole suppresses the Ca(2+)-activated translocation. No evidence for a role of the ATP-binding cassette transporter ABCA1 in the Ca(2+)-activated outward translocation is obtained. Suppression of phospholipid scrambling by dithioerythritol inhibits Ca(2+)-induced spheroechinocytosis and reduces the extent of subsequent microvesiculation. Scrambling of endogenous phospholipids is proposed to induce echinocytosis and to have only a stimulatory effect on microvesiculation.

Asbestos causes apoptosis in alveolar epithelial cells: role of iron-induced free radicals.

Asbestos causes asbestosis and malignancies by mechanisms that are not fully understood. Alveolar epithelial cell (AEC) injury by iron-induced reactive oxygen species (ROS) is one important mechanism. To determine whether asbestos causes apoptosis in AECs, we exposed WI-26 (human type I-like cells), A549 (human type II-like cells), and rat alveolar type II cells to amosite asbestos and assessed apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine-5'-triphosphate-biotin nick end labeling (TUNEL) staining, nuclear morphology, annexin V staining, DNA nucleosome formation, and caspase 3 activation. In contrast to control medium and TiO2, amosite asbestos and H2O2 each caused AEC apoptosis. A role for iron-catalyzed ROS was suggested by the finding that asbestos-induced AEC apoptosis and caspase 3 activation were each attenuated by either an iron chelator (phytic acid and deferoxamine) or a.OH scavenger (dimethyl-thiourea, salicylate, and sodium benzoate) but not by iron-loaded phytic acid. To determine whether asbestos causes apoptosis in vivo, rats received a single intratracheal instillation of amosite (5 mg) or normal saline solution, and apoptosis in epithelial cells in the bronchoalveolar duct regions was assessed by TUNEL staining. One week after exposure, amosite asbestos caused a 3-fold increase in the percentage of apoptotic cells in the bronchoalveolar duct regions as compared with control (control, 2.1% +/- 0.35%; asbestos, 7.61% +/- 0.15%; n = 3). However, by 4 weeks the number of apoptotic cells was similar to control. We conclude that asbestos-induced pulmonary toxicity may partly be caused by apoptosis in the lung epithelium that is mediated by iron-catalyzed ROS and caspase 3 activation.

Increased prevalence of gastroesophageal reflux symptoms in patients with COPD.

STUDY OBJECTIVES: To determine the prevalence of gastroesophageal reflux (GER) symptoms in patients with COPD and the association of GER symptoms with the severity of airways obstruction as assessed by pulmonary function tests (PFTs). DESIGN: Prospective questionnaire-based, cross-sectional analytic survey. SETTING: Outpatient pulmonary and general medicine clinics at a Veterans Administration hospital. PATIENTS: Patients with mild-to-severe COPD (n = 100) were defined based on American Thoracic Society criteria. The control group (n = 51) consisted of patients in the general medicine clinic without respiratory complaints or prior diagnosis of asthma or COPD. INTERVENTION: Both groups completed a modified version of the Mayo Clinic GER questionnaire. RESULTS: Compared to control subjects, a greater proportion of COPD patients had significant GER symptoms defined as heartburn and/or regurgitation once or more per week (19% vs 0%, respectively; p < 0.001), chronic cough (32% vs 16%; p = 0.03), and dysphagia (17% vs 4%; p = 0.02). Among patients with COPD and significant GER symptoms, 26% reported respiratory symptoms associated with reflux events, whereas control subjects denied an association. Significant GER symptoms were more prevalent in COPD patients with FEV(1) < or %, as compared with patients with FEV(1) > 50% of predicted (23% vs 9%, respectively; p = 0.08). In contrast, PFT results were similar among COPD patients with and without GER symptoms. An increased number of patients with COPD utilized antireflux medications, compared to control subjects (50% vs 27%, respectively; p = 0.008). CONCLUSIONS: The questionnaire demonstrated a higher prevalence of weekly GER symptoms in patients with COPD, as compared to control subjects. There was a trend toward higher prevalence of GER symptoms in patients with severe COPD; however, this difference did not reach statistical significance. We speculate that although GER may not worsen pulmonary function, greater expiratory airflow limitation may worsen GER symptoms in patients with COPD.

Cigarette smoke and asbestos activate poly-ADP-ribose polymerase in alveolar epithelial cells.

BACKGROUND: Cigarette smoke augments asbestos-induced bronchogenic carcinoma in a synergistic manner by mechanisms that are not established. One important mechanism may involve alveolar epithelial cell (AEC) injury resulting from oxidant-induced DNA damage that subsequently activates poly (ADP-ribose) polymerase (PARP), an enzyme involved in DNA repair that can deplete cellular energy stores. We previously showed that whole aqueous cigarette smoke extracts (CSE) augment amosite asbestos-induced DNA damage and cytotoxicity to cultured AEC in part by generating iron-induced free radicals. We hypothesized that CSE increase asbestos-induced AEC injury by triggering PARP activation resulting from DNA damage caused by iron-induced free radicals. METHODS: Aqueous CSE were prepared fresh on the day of each experiment. PARP activity in WI-26 (a type I-like cell line) and A549 (a type II-like cell line) cells was assessed by the uptake of labeled NAD over 4 hours and confirmed on the basis of the reduction of PARP levels in the presence of a PARP inhibitor, 3-aminobenzamide (3-ABA). Cell survival was assessed by trypan blue dye exclusion. RESULTS: Hydrogen peroxide (H2O2; 1-250 microM), CSE (0.4-10 vol%), and amosite asbestos (5-250 micrograms/cm2) each caused PARP activation in WI-26 and A549 cells. The combination of asbestos (5 micrograms/cm2) and CSE (0.04-10%) induced WI-26 and A549 cell PARP activation without evidence of synergism. 3-ABA significantly attenuated WI-26 and A549 cell PARP activity and cell death after exposure to H2O2, CSE, and asbestos. Phytic acid, an iron chelator, catalase, and superoxide dismutase each decreased WI-26 cell PARP activation caused by asbestos and CSE. CONCLUSIONS: CSE and asbestos induced PARP activation in cultured AEC in a nonsynergistic manner. These data provide further support that asbestos and cigarette smoke are genotoxic to relevant lung target cells and that iron-induced free radicals in part cause these effects.

Wetting characteristics of media emulating gastric fluids.

A variety of dissolution media have been used to simulate the physiological environment of the gastric region. The objective of this study was to formulate and examine the wetting properties of dispersions composed of the dominant surface active species found in the stomach at physiologically relevant concentrations. Systems representing the fed and fasted states were studied and compared to other media that have been considered for use as simulated gastric fluids. Dilute bile salt/phospholipid solutions and bile salt-lipid emulsions were formulated on the basis of available physiologic data to represent the fasted and fed states, respectively. Wetting was evaluated through the determination of the surface tension and contact angle of the various solutions using poly(methyl methacrylate) (PMMA) as a model surface. Additional surfactant solutions and other biorelevant media were also tested. Data were evaluated in terms of contact angle, surface tension and the thermodynamic stages of wetting. The results indicate that solutions patterned after the composition of the GI tract have significantly different wetting properties relative to the fed and fasted states. The surfactant solutions tested were significantly better wetting agents for the surface than the physiologically representative formulations. The implications for the formulation of surfactant-based biorelevant media are discussed.

Wetting behavior of bile salt-lipid dispersions and dissolution media patterned after intestinal fluids.

The wetting properties of bile salt-lipid solutions and dispersions patterned after the contents of the upper intestine in the fed and fasted states were evaluated using poly(methyl methacrylate) (PMMA) as a model substrate. The surface tension of the solutions and their contact angles on PMMA were measured. Media compositions for the intestinal fed and fasted states were estimated on the basis of physiologic data. The effect of various individual lipids and media composition was also evaluated relative to the adhesional, immersional, and spreading stages of wetting. In micellar systems, both the type and concentration of lipid present in the bile salt solution had an influence on wetting. The wetting of media patterned after gastrointestinal contents showed marked differences with respect to the fed or fasted state compositions. For the fed state compositions, alteration of the solution pH from 7.5 to 5.0 resulted in a significant change in wetting. The surface tension of the medium representative of the fasted state was 15 mN/m higher than that of the fed state. The wetting properties of the physiologically representative media formulated in this study were markedly different compared with other media proposed in the literature. Analysis of the wetting behavior of individual lipids as a function of concentration in bile salt solutions showed that they adsorb in a manner that progressively reduces the expected wetting of the surface. The results have implications for the design and formulation of both biorelevant and surfactant-based dissolution media.

Acceleration of phospholipid flip-flop in the erythrocyte membrane by detergents differing in polar head group and alkyl chain length.

The detergents, alkyltrimethylammonium bromide, N-alkyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (zwittergent), alkane sulfonate, alkylsulfate, alkyl-beta-D-glucopyranoside, alkyl-beta-D-maltoside, dodecanoyl-N-methylglucamide, polyethylene glycol monoalkyl ether and Triton X-100, all produce a concentration-dependent acceleration of the slow passive transbilayer movement of NBD-labeled phosphatidylcholine in the human erythrocyte membrane. Above a threshold concentration, which was well below the CMC and characteristic for each detergent, the flip rate increases exponentially upon an increase of the detergent concentration in the medium. The detergent-induced flip correlates with reported membrane-expanding effects of the detergents at antihemolytic concentrations. From the dependence of the detergent concentration required for a defined flip acceleration on the estimated membrane volume, membrane/water partition coefficients for the detergents could be determined and effective detergent concentrations in the membrane calculated. The effective membrane concentrations are similar for most types of detergents but are 10-fold lower for octaethylene glycol monoalkyl ether and Triton X-100. The effectiveness of a given type of detergent is rather independent of its alkyl chain length. Since detergents do not reduce the high temperature dependence of the flip process the detergent-induced flip is proposed to be due to an enhanced probability of formation of transient hydrophobic structural defects in the membrane barrier which may result from perturbation of the interfacial region of the bilayer by inserted detergent molecules.

Glutathione redox system in oxidative lung injury.

Glutathione (GSH) is a ubiquitous intracellular thiol present in all tissues, including lung. Besides maintaining cellular integrity by creating a reduced environment, GSH has multiple functions, including detoxification of xenobiotics, synthesis of proteins, nucleic acids, and leukotrienes. Present in high concentrations in bronchoalveolar lavage fluid (BALF), GSH provides protection to the lung from oxidative injury induced by different endogenous or exogenous pulmonary toxicants. Its depletion in the lung has been associated with the increased risk of lung damage and disease. The redox system of GSH consists of primary and secondary antioxidants, including glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), and glucose 6-phosphate dehydrogenase (G6PD). Alterations in the activities of these enzymes may reflect reduced cellular defense and may serve as surrogate markers of many lung diseases. As GSH is also involved in the regulation of expression of protooncogenes and apoptosis (programmed cell death), the development of diseases such as cancer and human immune deficiency may be affected by depleting or elevating cellular GSH levels. Exogenous delivery of GSH or its precursor N-acetyl cysteine (NAC) is being used as chemotherapeutic approach.

Cigarette smoke augments asbestos-induced alveolar epithelial cell injury: role of free radicals.

Cigarette smoke augments asbestos-induced bronchogenic carcinoma by mechanisms that are not established. Alveolar epithelial cell (AEC) injury due to oxidant-induced DNA damage and depletion of glutathione (GSH) and adenosine triphosphate (ATP) may be one important mechanism. We previously showed that amosite asbestos-induces hydroxyl radical production and DNA damage to cultured AEC and that phytic acid, an iron chelator, is protective. We hypothesized that whole cigarette smoke extracts (CSE) augment amosite asbestos-induced AEC injury by generating iron-induced free radicals that damage DNA and reduce cellular GSH and ATP levels. Asbestos or CSE each caused dose-dependent toxicity to AEC (WI-26 and rat alveolar type I-like cells) as assessed by 51chromium release. The combination of asbestos (5 microg/cm2) and CSE (0.O1-0.1%) caused synergistic injury whereas higher doses of each agent primarily had an additive toxic effect. Asbestos (5 microg/cm2) augmented CSE-induced (0.01-1.0%) AEC DNA damage over a 4 h exposure period as assessed by an alkaline unwinding, ethidium bromide fluorometric technique. These effects were synergistic in A549 cells and additive in WI-26 cells. Asbestos (5 microg/cm2) and CSE (0.5-1.0%) reduced A549 and WI-26 cell GSH levels as assessed spectrophotometrically and ATP levels as assessed by luciferin/luciferase chemiluminescence but a synergistic interaction was not detected. Phytic acid (500 microM) and catalase (100 microg/ml) each attenuated A549 cell DNA damage and depletion of ATP caused by asbestos and CSE. However, neither agent attenuated WI-26 cell DNA damage nor the reductions in GSH levels in WI-26 and A549 cells exposed to asbestos and CSE. We conclude that CSE enhance asbestos-induced DNA damage in cultured alveolar epithelial cells. These data provide additional support that asbestos and cigarette smoke are genotoxic to relevant target cells in the lung and that iron-induced free radicals may in part cause these effects.

Keratinocyte growth factor promotes alveolar epithelial cell DNA repair after H2O2 exposure.

Alveolar epithelial cell (AEC) injury and repair are important in the pathogenesis of oxidant-induced lung damage. Keratinocyte growth factor (KGF) prevents lung damage and mortality in animals exposed to various forms of oxidant stress, but the protective mechanisms are not yet established. Because DNA strand break (DNA-SB) formation is one of the earliest cellular changes that occurs after cells are exposed to an oxidant stress, we determined whether KGF reduces H2O2-induced pulmonary toxicity by attenuating AEC DNA damage. KGF (10-100 ng/ml) decreased H2O2 (0.05-0.5 mM)-induced DNA-SB formation in cultured A549 and rat alveolar type II cells measured by an alkaline unwinding, ethidium bromide fluorometric technique. The protective effects of KGF were independent of alterations in catalase, glutathione (GSH), or the expression of bcl-2 and bax, two protooncogenes known to regulate oxidant-induced apoptosis. Actinomycin D and cycloheximide abrogated protective effects of KGF. Furthermore, protection by KGF was completely blocked by 1) genistein, a tyrosine kinase inhibitor; 2) staurosporine and calphostin C, protein kinase C (PKC) inhibitors; and 3) aphidicolin, butylphenyl dGTP, and 2',3'-dideoxythymidine 5'-triphosphate, inhibitors of DNA polymerase. We conclude that KGF attenuates H2O2-induced DNA-SB formation in cultured AECs by mechanisms that involve tyrosine kinase, PKC, and DNA polymerases. These data suggest that the ability of KGF to protect against oxidant-induced lung injury is partly due to enhanced AEC DNA repair.

Evidence for a role of the multidrug resistance protein (MRP) in the outward translocation of NBD-phospholipids in the erythrocyte membrane.

Phosphatidylserine (PS) containing a 7-nitrobenz-2-oxa-1, 3-diazol-4-yl- (NBD-) hexanoyl residue, like native PS, preferentially distributes into the inner membrane leaflet of human erythrocytes. In the case of NBD-PS, this preference results from two opposite active processes, an inward translocation mediated by the aminophospholipid flippase and an outward translocation mediated by an ill-defined floppase. Selective inhibition of this floppase by alkylating reagents or cationic and anionic drugs increases the extent of accumulation of NBD-PS in the inner membrane leaflet from about 70% in control cells to about 90%. Different inhibitor sensitivities of the flippase and the floppase strongly suggest that both represent different entities. The floppase was characterized in further detail by comparing inhibitory effects of various compounds on this translocase with their effects on known primary active transport systems for amphiphilic compounds. The inhibitory effects of various drugs, glutathione conjugates and GSSG on the floppase activity closely correlate with those reported for the active transport by the multidrug resistance protein (MRP) while only poorly going parallel with those for the active transport by the low affinity pump for glutathione conjugates and the multidrug resistance MDR1 P-glycoprotein. The NBD-phospholipid floppase activity of the erythrocyte is thus probably a function of MRP.

Bacterial toxin-induced pulmonary epithelial cytotoxicity and the protective effect of dibutyryl-cAMP.

Bacterial infection is the most common cause of the adult respiratory distress syndrome which, in turn is associated with endothelial capillary permeability and alveolar oedema. Previously, we have demonstrated the direct cytotoxicity of the bacterial toxins Pseudomonas aeruginosa exotoxin A (Exo A) and Salmonella enteritidis lipopolysaccharide (LPS) on pulmonary endothelial cells. The purpose of this study was to investigate the effect of Exo A and LPS on pulmonary epithelial cells in vitro. We also tested the protective effect of dibutyryl cyclic adenosine monophosphate (db-cAMP) on Exo A-induced cytotoxicity. In cultured rat alveolar epithelial cells (RAEC) Exo A caused cytotoxicity as measured by 51Cr release from these cells. LPS did not injure RAEC's. Pretreatment of RAEC with db-cAMP (1 mM) attenuated Exo A induced cytotoxicity. We conclude that (1) Exo A directly injures epithelial lung cells and may contribute to lung injury in cases of bacterial infection; (2) db-cAMP protects alveolar epithelial cells against Exo A-induced cytotoxicity and (3) alveolar epithelial cells in this model are resistant to LPS induced injury.

Nonmediated flip-flop of anionic phospholipids and long-chain amphiphiles in the erythrocyte membrane depends on membrane potential.

The nonmediated inward translocation (flip) of the anionic fluorescent N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- (NBD-)labeled phospholipid phosphatidylmethanol (PM) from the outer to the inner membrane leaflet of human erythrocytes and vice versa depends on membrane potential. Interestingly, inside-positive potentials due to chloride gradients and the native chloride conductance of the cells resulted in an increase of the flip rates. This flip enhancement could be suppressed by addition of gramicidin D, which increases cation conductance, or 4,4'-diisothiocyanatostilbene-2,2'-disufonate (DIDS), which inhibits anion conductance. Conversely, inside negative potentials established by an outward-directed K+ gradient in the presence of gramicidin on DIDS-treated cells resulted in a decrease of flip rate. Flip rate exhibited an exponential dependence on membrane potential. The opposite effects of the positive and negative potentials were obtained for the outward translocation (flop) from the inner to the outer membrane leaflet. Similar potential dependencies were found for the nonmediated flip of anionic NBD-labeled phosphatidic acid (PA) and 2-(N-decyl)aminonaphthalene-6-sulfonic acid (2,6-DENSA) following blockage of the band-3-mediated component of flip. The membrane potential also influences the stationary distribution of the anionic lipids between the inner and outer leaflets. The distribution is shifted to the inner leaflet by increasingly positive potentials and to the outer leaflet by increasingly negative potentials. It is concluded that nonmediated flip-flop of the anionic phospholipids and the long-chain sulfonate represents electrogenic translocation of the unprotonated charged lipids across the hydrophobic barrier.

KGF facilitates repair of radiation-induced DNA damage in alveolar epithelial cells.

Administration of exogenous keratinocyte growth factor (KGF) prevents or attenuates several forms of oxidant-mediated lung injury. Because DNA damage in epithelial cells is a component of radiation pneumotoxicity, we determined whether KGF ameliorated DNA strand breaks in irradiated A549 cells. Cells were exposed to 137Cs gamma rays, and DNA damage was measured by alkaline unwinding and ethidium bromide fluorescence after a 30-min recovery period. Radiation induced a dose-dependent increase in DNA strand breaks. The percentage of double-stranded DNA after exposure to 30 Gy increased from 44.6 +/- 3.5% in untreated control cells to 61.6 +/- 5.0% in cells cultured with 100 ng/ml KGF for 24 h (P < 0.05). No reduction in DNA damage occurred when the cells were cultured with KGF but maintained at 0 degree C during and after irradiation. The sparing effect of KGF on radiation-induced DNA damage was blocked by aphidicolin, an inhibitor of DNA polymerases-alpha, -delta, and -epsilon and by butylphenyl dGTP, which blocks DNA polymerase-alpha strongly and polymerases-delta and -epsilon less effectively. However, dideoxythymidine triphosphate, a specific inhibitor of DNA polymerase-beta, did not abrogate the KGF effect. Thus KGF increases DNA repair capacity in irradiated pulmonary epithelial cells, an effect mediated at least in part by DNA polymerases-alpha, -delta, and -epsilon. Enhancement of DNA repair capability after cell damage may be one mechanism by which KGF is able to ameliorate oxidant-mediated alveolar epithelial injury.

Transbilayer reorientation of phospholipid probes in the human erythrocyte membrane. Lessons from studies on electroporated and resealed cells.

In order to characterize in more detail the previously observed (Dressler et al. (1983) Biochim. Biophys. Acta 732, 304-307) increases in transbilayer mobility of phospholipids in the erythrocyte membrane following electroporation at 0 degrees C and subsequent resealing at 37 degrees C of the cells, we have studied rates of flip and flop as well as steady state distributions of the fluorescent N-(NBD)-aminohexanoyl-analogues of the four major membrane phospholipids. Measurements comprised the passive non-mediated components as well as those mediated by specific translocases (flippase and floppase). The major new findings and insights can be summarized as follows. (1) The enhancement of passive transbilayer mobility which increases with the strength, duration, and number of field pulses at 0 degrees C, cannot be fully reversed by subsequent resealing at 37 degrees C. Flip-flop remains considerably elevated relative to the original values.(2) Enhanced mobilities induced by electroporation differ for the probes studied in the sequence SM <<< PS << PC < PE. Other membrane perturbations going along with enhanced flip-flop share only in part this pattern. (3) Mediated, ATP-dependent components of flip and flop of the probes are suppressed in electroporated/resealed cells, partly due to loss of cellular Mg2+, partly - in case of flippase - due to competition by externalized endogenous PS. (4) Electroporated/resealed cells provide an elegant means to demonstrate the contribution of various components of flip and flop to the steady state transbilayer distribution of phospholipids, in particular the role of passive mobility. The new, detailed information on the displacements of phospholipid between the two leaflets of the membrane bilayer in porated/resealed cells will help to understand erythrocyte shape changes following poration and during resealing (Henszen et al. (1993) Biol. Chem. Hoppe-Seyler 374, 114).