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J McCarthy1, X Gong2, D Nahirney2, M Duszyk2, MW Radomski11School of Pharmacy and Pharmaceutical Sciences, Panoz Institute, Trinity College Dublin, Dublin, Ireland; 2Department of Physiology, University of Alberta, Edmonton, Alberta, CanadaBackground: Over the last decade, nanotechnology has provided researchers with new nanometer materials, such as nanoparticles, which have the potential to provide new therapies for many lung diseases. In this study, we investigated the acute effects of polystyrene nanoparticles on epithelial ion channel function.Methods: Human submucosal Calu-3 cells that express cystic fibrosis transmembrane conductance regulator (CFTR) and baby hamster kidney cells engineered to express the wild-type CFTR gene were used to investigate the actions of negatively charged 20 nm polystyrene nanoparticles on short-circuit current in Calu-3 cells by Ussing chamber and single CFTR Cl- channels alone and in the presence of known CFTR channel activators by using baby hamster kidney cell patches.Results: Polystyrene nanoparticles caused sustained, repeatable, and concentration-dependent increases in short-circuit current. In turn, these short-circuit current responses were found to be biphasic in nature, ie, an initial peak followed by a plateau. EC50 values for peak and plateau short-circuit current responses were 1457 and 315.5 ng/mL, respectively. Short-circuit current was inhibited by diphenylamine-2-carboxylate, a CFTR Cl- channel blocker. Polystyrene nanoparticles activated basolateral K+ channels and affected Cl- and HCO3- secretion. The mechanism of short-circuit current activation by polystyrene nanoparticles was found to be largely dependent on calcium-dependent and cyclic nucleotide-dependent phosphorylation of CFTR Cl- channels. Recordings from isolated inside-out patches using baby hamster kidney cells confirmed the direct activation of CFTR Cl- channels by the nanoparticles.Conclusion: This is the first study to identify the activation of ion channels in airway cells after exposure to polystyrene-based nanomaterials. Thus, polystyrene nanoparticles cannot be considered as a simple neutral vehicle for drug delivery for the treatment of lung diseases, due to the fact that they may have the ability to affect epithelial cell function and physiological processes on their own.Keywords: CFTR, cystic fibrosis transmembrane conductance regulator, ion channels, K+ channels, lung cells, polystyrene nanoparticle

The effects of endogenous and exogenous nitric oxide (NO) on chloride currents were analyzed in A549 alveolar epithelial cells via whole-cell patch-clamp recordings. Whole-cell current measurements of epithelial ion transport in A549 alveolar epithelial cells indicated the dominant role of Cl- channels in generating membrane currents. Furthermore, the high permeability of Cl- indicated the role of non-cystic fibrosis conductance regulator Cl- channels in mediating chloride ion transport.

Mutations in polycystin-1, polycystin-2, or fibrocystin account for autosomal dominant or recessive polycystic kidney disease. Renal cystogenesis is linked to abnormal localization and function of these cystoproteins in renal primary cilia. They are also expressed in extrarenal tissues in which their functions are unclear. Here we found that human type-II alveolar epithelial A549, airway submucosal Calu-3 cells, and rat bronchioles contain primary or multiple cilia in which we detected these cystoproteins. At sub-confluency, polycystin-1 was expressed on plasma membrane, while polycystin-2 was localized to the ER of resting cells. Both polycystins were detected on the spindle and mid-body of mitotic cells, while fibrocystin was on centrosome throughout cell cycle. Polycystins and fibrocystin may participate in regulating mucociliary sensing and transport within pulmonary airways.

We demonstrate the effects on membrane of the tubulin-binding chemotherapy drugs: thiocolchicoside and taxol. Electrophysiology recordings across lipid membranes in aqueous phases containing drugs were used to investigate the drug effects on membrane conductance. Molecular dynamics simulation of the chemotherapy drug-lipid complexes was used to elucidate the mechanism at an atomistic level. Both drugs are observed to induce stable ion-flowing pores across membranes. Discrete pore current-time plots exhibit triangular conductance events in contrast to rectangular ones found for ion channels. Molecular dynamics simulations indicate that drugs and lipids experience electrostatic and van der Waals interactions for short periods of time when found within each other's proximity. The energies from these two interactions are found to be similar to the energies derived theoretically using the screened Coulomb and the van der Waals interactions between peptides and lipids due to mainly their charge properties while forming peptide-induced ion channels in lipid bilayers. Experimental and in silico studies together suggest that the chemotherapy drugs induce ion pores inside lipid membranes due to drug-lipid physical interactions. The findings reveal cytotoxic effects of drugs on the cell membrane, which may aid in novel drug development for treatment of cancer and other diseases., (© 2012 John Wiley & Sons A/S.)

Escherichia coli export the protein YebF into the extracellular medium by a two-step process. However, as no general outer membrane protein secretion system common to all E. coli strains has been reported, the mechanism of export has remained unclear. Herein, we identify the outer membrane proteins OmpF, OmpC, and OmpX as central to the YebF export mechanism using both genetic and planar lipid bilayer experiments. The nuclear magnetic resonance structural ensemble of YebF reveals a cystatin-like fold consisting of a structured core and an extended dynamic surface in a state of conformational exchange. This surface, conserved throughout YebF orthologs of Enterobacteriaceae, may facilitate the porin-mediated transport of YebF as amino acid substitutions of dynamic residues reduced secretion to the extracellular medium. Our results demonstrate that OmpF and OmpC not only operate to import ions and protein toxins but may also contribute to the export of the YebF protein family., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Protease-activated receptor 2 (PAR-2) is a G protein-coupled receptor possibly involved in the pathogenesis of asthma. PAR-2 also modulates ion transport in cultured epithelial cells, but these effects in native airways are controversial. The influence of allergic inflammation on PAR-2-induced changes in ion transport has received little attention. Here, we studied immediate changes in transepithelial short circuit current (I (sc)) induced by PAR-2 activation in the tracheas of naive and allergic mice. Activation of PAR-2 with an apically added activation peptide (AP) induced a small increase in I (sc), while a much larger increase was observed following basolateral AP addition. In ovalbumin-sensitized and -challenged animals used as a model of allergic airway inflammation, the effect of basolateral AP addition was enhanced. Responses to basolateral AP in both naive and allergic mice were not decreased by blocking sodium absorption with amiloride or CFTR function with CFTR(inh)172 but were reduced by the cyclooxygenase inhibitor indomethacin and largely blocked (>80%) by niflumic acid, a calcium-activated chloride channels' (CaCC) blocker. Allergic mice also showed an enhanced response to ATP and thapsigargin. There was no change in mRNA expression of Par-2 or of the chloride channels Ano1 (Tmem16a) and Bestrophin 2 in tracheas from allergic mice, while mRNA levels of Bestrophin 1 were increased. In conclusion, basolateral PAR-2 activation in the mouse airways led to increased anion secretion through apical CaCC, which was more pronounced in allergic animals. This could be a protective mechanism aimed at clearing allergens and defending against mucus plugging.

Asthma affects an estimated 300 million people worldwide and accounts for 1 of 250 deaths and 15 million disability-adjusted life years lost annually. Plastic-adherent bone marrow-derived cell (BMC) administration holds therapeutic promise in regenerative medicine. However, given the low cell engraftment in target organs, including the lung, cell replacement cannot solely account for the reported therapeutic benefits. This suggests that BMCs may act by secreting soluble factors. BMCs also possess antiinflammatory and immunomodulatory properties and may therefore be beneficial for asthma. Our objective was to investigate the therapeutic potential of BMC-secreted factors in murine asthma. In a model of acute and chronic asthma, intranasal instillation of BMC conditioned medium (CdM) prevented airway hyperresponsiveness (AHR) and inflammation. In the chronic asthma model, CdM prevented airway smooth muscle thickening and peribronchial inflammation while restoring blunted salbutamol-induced bronchodilation. CdM reduced lung levels of the T(H)2 inflammatory cytokines IL-4 and IL-13 and increased levels of IL-10. CdM up-regulated an IL-10-induced and IL-10-secreting subset of T regulatory lymphocytes and promoted IL-10 expression by lung macrophages. Adiponectin (APN), an antiinflammatory adipokine found in CdM, prevented AHR, airway smooth muscle thickening, and peribronchial inflammation, whereas the effect of CdM in which APN was neutralized or from APN knock-out mice was attenuated compared with wild-type CdM. Our study provides evidence that BMC-derived soluble factors prevent murine asthma and suggests APN as one of the protective factors. Further identification of BMC-derived factors may hold promise for novel approaches in the treatment of asthma.

Carnocyclin A (CclA) is a potent antimicrobial peptide from Carnobacterium maltaromaticum UAL307 that displays a broad spectrum of activity against numerous Gram-positive organisms. An amide bond links the N and C termini of this bacteriocin, imparting stability and structural integrity to this 60-amino acid peptide. CclA interacts with lipid bilayers in a voltage-dependent manner and forms anion selective pores. Several other circular bacteriocins have been reported, yet only one (enterocin AS-48) has been structurally characterized. We have now determined the solution structure of CclA by NMR and further examined its anion binding and membrane channel properties. The results reveal that CclA preferentially binds halide anions and has a structure that is surprisingly similar to that of AS-48 despite low sequence identity, different oligomeric state, and disparate function. CclA folds into a compact globular bundle, comprised of four helices surrounding a hydrophobic core. NMR studies show two fluoride ion binding modes for CclA. Our findings suggest that although other circular bacteriocins are likely to have diverse mechanisms of action, many may have a common structural motif. This shared three-dimensional arrangement resembles the fold of mammalian saposins, peptides that either directly lyse membranes or serve as activators of lipid-degrading enzymes.

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. In this study we have investigated the role of cholesterol in the adenosine-dependent regulation of ion transport in colonic epithelial cells. We observed that methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering molecule, enhanced adenosine A(2A) receptor-activated transepithelial short circuit current (I(sc)), but only from the basolateral side. Cholesterol is a major constituent of membrane microdomains, called lipid rafts that also contain sphingolipids. However, studies with the sphingomyelin-degrading enzyme, sphingomyelinase, and the cholesterol-binding agent, filipin, indicated that the change in the level of cholesterol alone was sufficient to control the adenosine-modulated I(sc). Cholesterol depletion had a major effect on the functional selectivity of A(2A) receptors. Under control conditions, adenosine activated I(sc) more potently than the specific A(2A) agonist, CGS-21680, and the current was inhibited by XE991, an inhibitor of cAMP-dependent K(+) channels. Following cholesterol depletion, CGS-21680 activated I(sc) more potently than adenosine, and the current was inhibited by clotrimazole, an inhibitor of Ca(2+)-activated K(+) (IK1) channels. Co-immunoprecipitation experiments revealed that A(2A) receptors associate with IK1 channels following cholesterol depletion. These results suggest that cholesterol content in colonic epithelia affects adenosine-mediated anion secretion by controlling agonist-selective signaling.

Bacterial resistance to conventional antibiotics is a major challenge in controlling infectious diseases and has necessitated the development of novel approaches in antimicrobial therapy. One such approach is the use of antimicrobial peptides, such as the bacterially produced bacteriocins. Carnocyclin A (CclA) is a 60-amino acid circular bacteriocin produced by Carnobacterium maltaromaticum UAL307 that exhibits potent activity against many Gram-positive bacteria. Lipid bilayer and single channel recording techniques were applied to study the molecular mechanisms by which CclA interacts with the lipid membrane and exerts its antimicrobial effects. Here we show that CclA can form ion channels with a conductance of 35 pS in 150 mM NaCl solution. This channel displays a linear current-voltage relationship, is anion-selective, and its activation is strongly voltage-dependent. The formation of ion channels by CclA is driven by the presence of a negative membrane potential and may result in dissipation of membrane potential. Carnocyclin A's unique functional activities as well as its circular structure make it a potential candidate for developing novel antimicrobial drugs.

Cystic fibrosis (CF) is caused by the mutation in CF transmembrane conductance regulator (CFTR), a cAMP-dependent Cl(-) channel at the plasma membrane of epithelium. The most common mutant, DeltaF508 CFTR, has competent Cl(-) channel function, but fails to express at the plasma membrane since it is retained in the endoplasmic reticulum (ER) by the ER quality control system. Here, we show that calnexin (CNX) is not necessary for the ER retention of DeltaF508 CFTR. Our data show that CNX knockout (KO) does not affect the biosynthetic processing, cellular localization or the Cl(-) channel function of DeltaF508 CFTR. Importantly, cAMP-induced Cl(-) current in colonic epithelium from CNX KO/DeltaF508 CFTR mice was comparable with that of DeltaF508 CFTR mice, indicating that CNX KO failed to rescue the ER retention of DeltaF508 CFTR in vivo. Moreover, we show that CNX assures the efficient expression of WT CFTR, but not DeltaF508 CFTR, by inhibiting the proteasomal degradation, indicating that CNX might stimulate the productive folding of WT CFTR, but not DeltaF508 CFTR, which has folding defects.

Butyrate and other short-chain fatty acids (SCFA), produced by colonic bacterial flora, affect numerous epithelial cell functions. To better understand how SCFA regulate ion transport, we investigated the effects of 4-phenylbutyrate (4-PBA) on Na(+) absorption in T84 cells. Under standard cell culture conditions, the short circuit current did not display any amiloride-sensitive Na(+) absorption and was wholly representative of Cl(-) secretion. However, when T84 cells were grown in the presence of 5 mM 4-PBA, a gradual appearance of amiloride-sensitive Na(+) channel (ENaC) activity was observed that reached a plateau after 24 h. Quantitative RT-PCR and Western blot studies of ENaC subunit expression indicated that 4-PBA stimulated alpha and gamma subunits. Trichostatin A, an inhibitor of histone deacetylase, mimicked the effects of 4-PBA, suggesting that 4-PBA affects ENaC expression by inhibiting deacetylases. 4-PBA had no effect on ENaC expression in airway epithelial cells indicating tissue-specific effect. We conclude that butyrate plays an important role in regulating colonic Na(+) absorption by increasing ENaC transcription and activity.

Mast cells' hyperplasia and activation are prominent features in Trichinella spiralis infection. Recently, it was shown that TSL-1 antigens from T. spiralis muscle larvae induce IL-4 and TNF release by unsensitized, normal mast cells (MC) involving an Ig-independent mechanism. In this study, we characterized histamine secretion induced by TSL-1 antigens from normal, unsensitized rat peritoneal MC. Maximum histamine secretion (30+/-5.3% SEM, n=13) was achieved with 30 ng/mL TSL-1 antigens. However, TSL-1 did not induce an increase in beta-hexosaminidase release or NADPH oxidase activity by MC. Interestingly, histamine secretion by TSL-1 was completed at 10s, and was inhibited by both Bordetella pertussis toxin and neuraminidase V, characteristics similar to those involved in substance P-induced histamine secretion. However, in contrast to substance P, TSL-1 induced histamine secretion in the absence of detectable changes in intracellular Ca(2+). We are investigating the molecular pathways involved in MC activation by TSL-1.

The presence of basolateral Cl(-) channels in airway epithelium has been reported in several studies, but little is known about their role in the regulation of anion secretion. The purpose of this study was to characterize regulation of these channels by nitric oxide (NO) in Calu-3 cells. Transepithelial measurements revealed that NO donors activated a basolateral Cl(-) conductance sensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and anthracene-9-carboxylic acid. Apical membrane permeabilization studies confirmed the basolateral localization of NO-activated Cl(-) channels. Experiments using 8-bromo cyclic guanosine monophosphate (8Br-cGMP) and selective inhibitors of soluble guanylyl cyclase and inducible NO synthase (1H-[1, 2, 4] oxadiazolol-[4, 3-a] quinoxalin-1-one [ODQ] and 1400W [N-(3-Aminomethyl)benzyl)acetamidine], respectively) demonstrated that NO activated Cl(-) channels via a cGMP-dependent pathway. Anion replacement and (36)Cl(-) flux studies showed that NO affected both Cl(-) and HCO (3) (-) secretion. Two different types of Cl(-) channels are known to be present in the basolateral membrane of epithelial cells: Zn(2+)-sensitive ClC-2 and DIDS-sensitive bestrophin channels. S-Nitrosoglutathione (GSNO) activated Cl(-) conductance in the presence of Zn(2+) ions, indicating that ClC-2 channel function was not affected by GSNO. In contrast, DIDS completely inhibited GSNO-activated Cl(-) conductance. Bestrophin immunoprecipitation studies showed that under control conditions bestrophin channels were not phosphorylated but became phosphorylated after GSNO treatment. The presence of bestrophin in airway epithelia was confirmed using immunohistochemistry. We conclude that basolateral Cl(-) channels play a major role in the NO-dependent regulation of anion secretion in Calu-3 cells.