Mass Spectrometric Analysis of the Active Site Tryptic Peptide of Recombinant O6-Methylguanine-DNA Methyltransferase Following Incubation with Human Colorectal DNA Reveals the Presence of an O6-Alkylguanine Adductome.

Human exposure to DNA alkylating agents is poorly characterized, partly because only a limited range of specific alkyl DNA adducts have been quantified. The human DNA repair protein, O6-methylguanine O6-methyltransferase (MGMT), irreversibly transfers the alkyl group from DNA O6-alkylguanines (O6-alkGs) to an acceptor cysteine, allowing the simultaneous detection of multiple O6-alkG modifications in DNA by mass spectrometric analysis of the MGMT active site peptide (ASP). Recombinant MGMT was incubated with oligodeoxyribonucleotides (ODNs) containing different O6-alkGs, Temozolomide-methylated calf thymus DNA (Me-CT-DNA), or human colorectal DNA of known O6-MethylG (O6-MeG) levels. It was digested with trypsin, and ASPs were detected and quantified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. ASPs containing S-methyl, S-ethyl, S-propyl, S-hydroxyethyl, S-carboxymethyl, S-benzyl, and S-pyridyloxobutyl cysteine groups were detected by incubating MGMT with ODNs containing the corresponding O6-alkGs. The LOQ of ASPs containing S-methylcysteine detected after MGMT incubation with Me-CT-DNA was <0.05 pmol O6-MeG per mg CT-DNA. Incubation of MGMT with human colorectal DNA produced ASPs containing S-methylcysteine at levels that correlated with those of O6-MeG determined previously by HPLC-radioimmunoassay (r2 = 0.74; p = 0.014). O6-CMG, a putative O6-hydroxyethylG adduct, and other potential unidentified MGMT substrates were also detected in human DNA samples. This novel approach to the identification and quantitation of O6-alkGs in human DNA has revealed the existence of a human DNA alkyl adductome that remains to be fully characterized. The methodology establishes a platform for characterizing the human DNA O6-alkG adductome and, given the mutagenic potential of O6-alkGs, can provide mechanistic information about cancer pathogenesis.

For Better and For Worse: Frequent Gamblers Use Dual Counterfactuals to Justify Continued Gambling.

How might frequent gamblers convince themselves to keep playing despite persistent losses or after a win that should be savored? The purpose of this research is to examine the unexplored question of how frequent gamblers' use counterfactual thinking to motivate their desire to continue gambling. Using a sample of n = 69 high and n = 69 low frequency gamblers in a field setting, we found that infrequent gamblers tended to consider how the perceived outcome of losing "could have been better" (i.e., upward counterfactual thinking), and how a winning outcome "could have been worse" (i.e., downward counterfactual thinking). This pattern of counterfactual thinking is considered typical in many settings and may, in a gambling context, support a potentially more responsible approach by helping infrequent gamblers to learn from past mistakes to avoid significant future losses and to savor wins to protect returns gained. Alternatively, we found that frequent gamblers were more likely to generate 'dual counterfactuals' which include both upward and downward counterfactuals in response to losses and wins. We argue that this dual pattern of counterfactual thinking may allow frequent gamblers to more easily justify their desire to continue gambling. Findings suggest that challenging gamblers counterfactual thinking patterns could assist clinicians in moderating the potential for high-risk behaviors.

Functionally additive fixed positive and negative charges in the CFTR channel pore control anion binding and conductance.

Ion channels use charged amino-acid residues to attract oppositely charged permeant ions into the channel pore. In the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, a number of arginine and lysine residues have been shown to be important for Cl- permeation. Among these, two in close proximity in the pore-Lys95 and Arg134-are indispensable for anion binding and high Cl- conductance, suggesting that high positive charge density is required for pore function. Here we used mutagenesis and functional characterization to show that a nearby pore-lining negatively charged residue (Glu92) plays a functionally additive role with these two positive charges. While neutralization of this negative charge had little effect on anion binding or Cl- conductance, such neutralization was able to reverse the detrimental effects of removing the positive charge at either Lys95 or Arg134, as well as the similar effects of introducing a negative charge at a neighboring residue (Ser1141). Furthermore, neutralization of Glu92 greatly increased the susceptibility of the channel to blockage by divalent S2O32- anions, mimicking the effect of introducing additional positive charge in this region; this effect was reversed by concurrent neutralization of either Lys95 or Arg134. Across a panel of mutant channels that introduced or removed fixed charges at these four positions, we found that many pore properties are dependent on the overall charge or charge density. We propose that the CFTR pore uses a combination of positively and negatively charged residues to optimize the anion binding and Cl- conductance properties of the channel.

Two positively charged amino acid side-chains in the inner vestibule of the CFTR channel pore play analogous roles in controlling anion binding and anion conductance.

Positively charged amino acid side-chains play important roles in anion binding and permeation through the CFTR chloride channel. One pore-lining lysine residue in particular (K95) has been shown to be indispensable for anion binding, conductance, and selectivity. Here, we use functional investigation of CFTR to show that a nearby arginine (R134) plays a functionally analogous role. Removal of this positive charge (in the R134Q mutant) drastically reduces single-channel conductance, weakens binding of both permeant and blocking anions, and abolishes the normal anion conductance selectivity pattern. Each of these functional effects was reversed by a second-site mutation (S1141K) that introduces an ectopic positive charge to a nearby pore-lining residue. Substituted cysteine accessibility experiments confirm that R134-but not nearby residues in the same transmembrane helix-is accessible within the pore lumen. These results suggest that K95 and R134, which are very close together within the inner vestibule of the pore, play analogous, important roles, and that both are required for the normal anion binding and anion conductance properties of the pore. Nevertheless, that fact that both positive charges can be "transplanted" to other sites in the inner vestibule with little effect on channel permeation properties indicates that it is the overall number of charges-rather than their exact locations-that controls pore function.

Electrostatic Tuning of Anion Attraction from the Cytoplasm to the Pore of the CFTR Chloride Channel.

Anions enter from the cytoplasm into the channel pore of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel not via a central pathway but via a single lateral portal or fenestration. High Cl- conductance is dependent on electrostatic attraction of cytoplasmic Cl- ions by four positively charged amino acid side-chains located within this portal. Here we use a mutagenic approach to investigate the functional effects of transplanting or supplementing these positive charges at nearby portal-lining sites. Using patch clamp recording, we find that the functionally important positive charges at K190 and R303 can be transplanted to four nearby sites (N186, L197, W356, and A367) with little loss of Cl- conductance. Introduction of additional positive charge at these sites had almost no effect on Cl- conductance, but did increase the sensitivity to channel block by intracellular suramin and Pt(NO2)42- anions. We suggest that it is the number of positive charges within the portal, rather than their exact location, that is the most important factor influencing Cl- conductance. The portal appears well optimized in terms of charge distribution to maximize Cl- conductance.

Contribution of the eighth transmembrane segment to the function of the CFTR chloride channel pore.

Our molecular understanding of the cystic fibrosis transmembrane conductance regulator (CFTR)-the chloride channel that is mutated in cystic fibrosis-has been greatly enhanced by a number of recent atomic-level structures of the protein in different conformations. One surprising aspect of these structures was the finding that the eighth of CFTR's 12 membrane-spanning segments (TM8) appeared close to the channel pore. Although functional evidence supports a role for other TMs in forming the pore, such a role for TM8 has not previously been reported. Here, we use patch-clamp recording to investigate the functional role of TM8. Using substituted cysteine accessibility mutagenesis, we find that three amino acid side-chains in TM8 (Y913, Y914, and Y917) are exposed to the extracellular, but not the intracellular, solution. Cysteine cross-linking experiments suggest that Y914 and Y917 are in close proximity to L102 (TM1) and F337 (TM6), respectively, suggesting that TM8 contributes to the narrow selectivity filter region of the pore. Different amino acid substitutions suggest that Y914, and to a lesser extent Y917, play important roles in controlling anion flux through the open channel. Furthermore, substitutions that reduce side-chain volume at Y917 severely affect channel gating, resulting in a channel with an extremely unstable open state. Our results suggest that pore-lining TM8 is among the most important TMs controlling the permeation phenotype of the CFTR channel, and also that movement of TM8 may be critically involved in channel gating.

Pomegranate Juice Supplementation Alters Utero-Placental Vascular Function and Fetal Growth in the eNOS-/- Mouse Model of Fetal Growth Restriction.

The eNOS-/- mouse provides a well-characterized model of fetal growth restriction (FGR) with altered uterine and umbilical artery function and reduced utero- and feto-placental blood flow. Pomegranate juice (PJ), which is rich in antioxidants and bioactive polyphenols, has been posited as a beneficial dietary supplement to promote cardiovascular health. We hypothesized that maternal supplementation with PJ will improve uterine and umbilical artery function and thereby enhance fetal growth in the eNOS-/- mouse model of FGR. Wild type (WT, C57Bl/6J) and eNOS-/- mice were supplemented from E12.5-18.5 with either PJ in their drinking water or water alone. At E18.5 uterine (UtA) and umbilical (UmbA) arteries were isolated for study of vascular function, fetuses and placentas were weighed and fetal biometric measurements taken. PJ supplementation significantly increased UtA basal tone (both genotypes) and enhanced phenylephrine-induced contraction in eNOS-/- but not WT mice. Conversely PJ significantly reduced UtA relaxation in response to both acetylcholine (Ach) and sodium nitroprusside (SNP), endothelium dependent and independent vasodilators respectively from WT but not eNOS-/- mice. UmbA sensitivity to U46619-mediated contraction was increased by PJ supplementation in WT mice; PJ enhanced contraction and relaxation of UmbA to Ach and SNP respectively in both genotypes. Contrary to our hypothesis, the changes in artery function induced by PJ were not associated with an increase in fetal weight. However, PJ supplementation reduced litter size and fetal abdominal and head circumference in both genotypes. Collectively the data do not support maternal PJ supplementation as a safe or effective treatment for FGR.

Conformational change of the extracellular parts of the CFTR protein during channel gating.

Cystic fibrosis can be treated by potentiators, drugs that interact directly with the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel to increase its open probability. These substances likely target key conformational changes occurring during channel opening and closing, however, the molecular bases of these conformational changes, and their susceptibility to manipulation are poorly understood. We have used patch clamp recording to identify changes in the three-dimensional organization of the extracellularly accessible parts of the CFTR protein during channel opening and closing. State-dependent formation of both disulfide bonds and Cd2+ bridges occurred for pairs of cysteine side-chains introduced into the extreme extracellular ends of transmembrane helices (TMs) 1, 6, and 12. Between each of these three TMs, we found that both disulfide bonds and metal bridges formed preferentially or exclusively in the closed state and that these inter-TM cross-links stabilized the closed state. These results indicate that the extracellular ends of these TMs are close together when the channel is closed and that they separate from each other when the channel opens. These findings identify for the first time key conformational changes in the extracellular parts of the CFTR protein that can potentially be manipulated to control channel activity.

Functional organization of cytoplasmic portals controlling access to the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel pore.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that apparently has evolved from an ancestral active transporter. Key to the CFTR's switch from pump to channel function may have been the appearance of one or more "lateral portals." Such portals connect the cytoplasm to the transmembrane channel pore, allowing a continuous pathway for the electrodiffusional movement of Cl- ions. However, these portals remain the least well-characterized part of the Cl- transport pathway; even the number of functional portals is uncertain, and if multiple portals do exist, their relative functional contributions are unknown. Here, we used patch-clamp recording to identify the contributions of positively charged amino acid side chains located in CFTR's cytoplasmic transmembrane extensions to portal function. Mutagenesis-mediated neutralization of several charged side chains reduced single-channel Cl- conductance. However, these same mutations differentially affected channel blockade by cytoplasmic suramin and Pt(NO2)42- anions. We considered and tested several models by which the contribution of these positively charged side chains to one or more independent or non-independent portals to the pore could affect Cl- conductance and interactions with blockers. Overall, our results suggest the existence of a single portal that is lined by several positively charged side chains that interact electrostatically with both Cl- and blocking anions. We further propose that mutations at other sites indirectly alter the function of this single portal. Comparison of our functional results with recent structural information on CFTR completes our picture of the overall molecular architecture of the Cl- permeation pathway.

Exposure to omentum adipose tissue conditioned medium from obese pregnant women promotes myometrial artery dysfunction.

AIM: Underlying mechanisms of poor pregnancy outcome in obese (OB) mothers (body mass index [BMI] ≥ 30 kg/m2 ) are unknown. Our studies demonstrate that OB pregnant women have altered myometrial artery (MA) function related to the thromboxane and nitric oxide pathways. In obesity, increased central fat mass is associated with an altered endocrine milieu. We tested the hypothesis that in OB pregnant women the omentum, a central fat store, releases factors that promote dysfunction in normal MAs. METHODS: Myometrial and omental adipose tissue biopsies were obtained from women with uncomplicated term pregnancies. Omental adipose tissue explants from six normal weight (NW; BMI 18.5-24.9 kg/m2 ) and six OB (BMI ≥ 30 kg/m2 ) women were cultured and the conditioned medium collected and pooled to produce NW medium and OB medium. Adipokine concentrations were measured using enzyme-linked immunosorbent assays. Wire myography was used to assess the effect of conditioned medium (NW or OB; N = 7) or leptin (100 nM; N = 5) exposure on MA responses to U46619 (thromboxane-mimetic) and bradykinin (endothelial-dependent vasodilator). RESULTS: OB medium had higher leptin and lower adiponectin levels than NW medium. U46619 and bradykinin concentration response curves shifted upwards in MAs exposed to OB medium but were unaffected by leptin. CONCLUSIONS: Omental adipose tissue from OB pregnant women produced altered concentrations of adipokines. Acute OB medium exposure induced MA dysfunction, an effect not mirrored by exposure to leptin. These data suggest that an aberrant endocrine environment created by increased central adiposity in OB pregnant women induces vascular endothelial dysregulation, which may predispose them to a poor pregnancy outcome.

Vitamin D attenuates sphingosine-1-phosphate (S1P)-mediated inhibition of extravillous trophoblast migration.

INTRODUCTION: Failure of trophoblast invasion and remodelling of maternal blood vessels leads to the pregnancy complication pre-eclampsia (PE). In other systems, the sphingolipid, sphingosine-1-phosphate (S1P), controls cell migration therefore this study determined its effect on extravillous trophoblast (EVT) function. METHODS: A transwell migration system was used to assess the behaviour of three trophoblast cell lines, Swan-71, SGHPL-4, and JEG3, and primary human trophoblasts in the presence or absence of S1P, S1P pathway inhibitors and 1,25(OH)2D3. QPCR and immunolocalisation were used to demonstrate EVT S1P receptor expression. RESULTS: EVTs express S1P receptors 1, 2 and 3. S1P inhibited EVT migration. This effect was abolished in the presence of the specific S1PR2 inhibitor, JTE-013 (p < 0.05 versus S1P alone) whereas treatment with the S1R1/3 inhibitor, FTY720, had no effect. In other cell types S1PR2 is regulated by vitamin D; here we found that treatment with 1,25(OH)2D3 for 48 or 72 h reduces S1PR2 (4-fold; <0.05), but not R1 and R3, expression. Moreover, S1P did not inhibit the migration of cells exposed to 1,25(OH)2D3 (p < 0.05). DISCUSSION: This study demonstrates that although EVT express three S1P receptor isoforms, S1P predominantly signals through S1PR2/Gα12/13 to activate Rho and thereby acts as potent inhibitor of EVT migration. Importantly, expression of S1PR2, and therefore S1P function, can be down-regulated by vitamin D. Our data suggest that vitamin D deficiency, which is known to be associated with PE, may contribute to the impaired trophoblast migration that underlies this condition.

Contribution of a leucine residue in the first transmembrane segment to the selectivity filter region in the CFTR chloride channel.

The anion selectivity and conductance of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are determined predominantly by interactions between permeant anions and the narrow region of the channel pore. This narrow region has therefore been described as functioning as the "selectivity filter" of the channel. Multiple pore-lining transmembrane segments (TMs) have previously been shown to contribute to the selectivity filter region. However, little is known about the three-dimensional organization of this region, or how multiple TMs combine to determine its functional properties. In the present study we have used patch clamp recording to identify changes in channel function associated with the formation of disulfide cross-links between cysteine residues introduced into different TMs within the selectivity filter. Cysteine introduced at position L102 in TM1 was able to form disulfide bonds with F337C and T338C in TM6, two positions that are known to play key roles in determining anion permeation properties. Consistent with this proximal arrangement of L102, F337 and T338, different mutations at L102 altered anion selectivity and conductance properties in a way that suggests that this residue plays an important role in determining selectivity filter function, albeit a much lesser role than that of F337. These results suggest an asymmetric three-dimensional arrangement of the key selectivity filter region of the pore, as well as having important implications regarding the molecular mechanism of anion permeation.

The atrial natriuretic peptide (ANP) knockout mouse does not exhibit the phenotypic features of pre-eclampsia or demonstrate fetal growth restriction.

The ANP knockout mouse is reported to exhibit pregnancy-associated hypertension, proteinuria and impaired placental trophoblast invasion and spiral artery remodeling, key features of pre-eclampsia (PE). We hypothesized that these mice may provide a relevant model of human PE with associated fetal growth restriction (FGR). Here, we investigated pregnancies of ANP wild type (ANP(+/+)), heterozygous (ANP(+/-)) and knockout (ANP(-/-)) mice. Maternal blood pressure did not differ between genotypes (E12.5, E17.5), and fetal weight (E18.5) was unaffected. Placental weight was greater in ANP(-/-) versus ANP(+/+) mice. Therefore, in our hands, the ANP model does not express phenotypic features of PE with FGR.

Breaks in Play: Do They Achieve Intended Aims?

Breaks in play represent a responsible gambling strategy designed to disrupt states of dissociation and enhance the likelihood of drawing attention to a player's session behaviour and expenditure with respect to time and money. The aim of the break in play is to motivate the player to modify or cease gambling so the activity remains within affordable levels. The aim of this study was to investigate whether imposed breaks in play in the absence of accompanying warning messages were effective in reducing cravings. Participants (141 university students) were randomly allocated to one of three conditions: 15 min computer simulated Black Jack play followed by no break, a 3 or 8 min break in play. Participants were administered a battery of measures to assess problem gambling card play, cravings, and dissociation to assess the effects of length of break on cravings. Results indicated that cravings increased rather than decreased with imposed breaks in play, and that the strength of cravings were higher following the eight- compared to 3-min break. It was concluded that breaks in play in isolation might produce counterproductive, unintended, and even perverse effects. The policy implications for responsible gambling strategies is that breaks in play ought to be accompanied with warning and/or personal appraisal messages if optimal effects in reducing within session gambling expenditure are to be achieved.

Mast cells protect against Pseudomonas aeruginosa-induced lung injury.

Pseudomonas aeruginosa, an opportunistic pathogen, is the leading cause of morbidity and mortality in immune-compromised individuals. Maintaining the integrity of the respiratory epithelium is critical for an effective host response to P. aeruginosa. Given the close spatial relationship between mast cells and the respiratory epithelium, and the importance of tightly regulated epithelial permeability during lung infections, we examined whether mast cells influence airway epithelial integrity during P. aeruginosa lung infection in a mouse model. We found that mast cell-deficient Kit(W-sh)/Kit(W-sh) mice displayed greatly increased epithelial permeability, bacterial dissemination, and neutrophil accumulation compared with wild-type animals after P. aeruginosa infection; these defects were corrected on reconstitution with mast cells. An in vitro Transwell co-culture model further demonstrated that a secreted mast cell factor decreased epithelial cell apoptosis and tumor necrosis factor production after P. aeruginosa infection. Together, our data demonstrate a previously unrecognized role for mast cells in the maintenance of epithelial integrity during P. aeruginosa infection, through a mechanism that likely involves prevention of epithelial apoptosis and tumor necrosis factor production. Our understanding of mechanisms of the host response to P. aeruginosa will open new avenues for the development of successful preventative and treatment strategies.

Maternal obesity impairs specific regulatory pathways in human myometrial arteries.

Obese women (body mass index ≥30 kg/m(2)) are at greater risk than normal weight women of pregnancy complications associated with maternal and infant morbidity, particularly the development of cardiovascular disease and metabolic disorders in later life; why this occurs is unknown. Nonpregnant, obese individuals exhibit systemic vascular endothelial dysfunction. We tested the hypothesis that obese pregnant women have altered myometrial arterial function compared to pregnant women of normal (18-24 kg/m(2)) and overweight (25-29 kg/m(2)) body mass index. Responses to vasoconstrictors, U46619 (thromboxane mimetic) and arginine vasopressin, and vasodilators, bradykinin and the nitric oxide donor sodium nitroprusside, were assessed by wire myography in myometrial arteries from normal weight (n = 18), overweight (n = 18), and obese (n = 20) women with uncomplicated pregnancies. Thromboxane-prostanoid receptor expression was assessed using immunostaining in myometrial arteries of normal weight and obese women. Vasoconstriction and vasodilatation were impaired in myometrial arteries from obese women with otherwise uncomplicated pregnancies. Disparate agonist responses suggest that vascular function in obese women is not globally dysregulated but may be specific to thromboxane and nitric oxide pathways. Because obesity rates are escalating, it is important to identify the mechanisms underlying impaired vascular function and establish why some obese women compensate for vascular dysfunction and some do not. Future studies are needed to determine whether central adiposity results in an altered endocrine milieu that may promote vascular dysfunction by altering the function of perivascular adipose tissue.

Sildenafil citrate increases fetal weight in a mouse model of fetal growth restriction with a normal vascular phenotype.

Fetal growth restriction (FGR) is defined as the inability of a fetus to achieve its genetic growth potential and is associated with a significantly increased risk of morbidity and mortality. Clinically, FGR is diagnosed as a fetus falling below the 5(th) centile of customised growth charts. Sildenafil citrate (SC, Viagra™), a potent and selective phosphodiesterase-5 inhibitor, corrects ex vivo placental vascular dysfunction in FGR, demonstrating potential as a therapy for this condition. However, many FGR cases present without an abnormal vascular phenotype, as assessed by Doppler measures of uterine/umbilical artery blood flow velocity. Thus, we hypothesized that SC would not increase fetal growth in a mouse model of FGR, the placental-specific Igf2 knockout mouse, which has altered placental exchange capacity but normal placental blood flow. Fetal weights were increased (by 8%) in P0 mice following maternal SC treatment (0.4 mg/ml) via drinking water. There was also a trend towards increased placental weight in treated P0 mice (P = 0.056). Additionally, 75% of the P0 fetal weights were below the 5(th) centile, the criterion used to define human FGR, of the non-treated WT fetal weights; this was reduced to 51% when dams were treated with SC. Umbilical artery and vein blood flow velocity measures confirmed the lack of an abnormal vascular phenotype in the P0 mouse; and were unaffected by SC treatment. (14)C-methylaminoisobutyric acid transfer (measured to assess effects on placental nutrient transporter activity) per g placenta was unaffected by SC, versus untreated, though total transfer was increased, commensurate with the trend towards larger placentas in this group. These data suggest that SC may improve fetal growth even in the absence of an abnormal placental blood flow, potentially affording use in multiple sub-populations of individuals presenting with FGR.

Organic cation transporters in human nasal primary culture: expression and functional activity.

BACKGROUND: The majority of drugs cross epithelial cells by either passive diffusion or via carrier-mediated drug transporters. The aim of this study was to investigate the transport characteristics, protein expression and localization of organic cation transporters in human nasal epithelium. METHODS & RESULTS: The expression, localization and transport characteristics of the transporters were investigated using permeation, PCR and immunohistochemistry. The uptake of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide followed Michaelis-Menten kinetics. Its intracellular accumulation of the compound was inhibited by organic cation transporters (OCTs) and carnitine/organic cation transporter (OCTNs) inhibitors. Detected OCT1-3, OCTN1 and OCTN2 gene transcripts correlated with immunohistological staining for OCT1-3, OCTN1 and OCTN2 antibodies. Except for OCTN1, the antibodies were generally localized on the apical side of the epithelial cells. CONCLUSION: Based on the immunohistochemical and uptake/transport studies, we conclude that the human nasal epithelium expresses OCT1-3, OCTN1 and OCTN2 transporters mainly on the apical side of the nasal cells.

Development and characterization of a 3D multicell microtissue culture model of airway smooth muscle.

Airway smooth muscle (ASM) cellular and molecular biology is typically studied with single-cell cultures grown on flat 2D substrates. However, cells in vivo exist as part of complex 3D structures, and it is well established in other cell types that altering substrate geometry exerts potent effects on phenotype and function. These factors may be especially relevant to asthma, a disease characterized by structural remodeling of the airway wall, and highlights a need for more physiologically relevant models of ASM function. We utilized a tissue engineering platform known as microfabricated tissue gauges to develop a 3D culture model of ASM featuring arrays of ∼0.4 mm long, ∼350 cell "microtissues" capable of simultaneous contractile force measurement and cell-level microscopy. ASM-only microtissues generated baseline tension, exhibited strong cellular organization, and developed actin stress fibers, but lost structural integrity and dissociated from the cantilevers within 3 days. Addition of 3T3-fibroblasts dramatically improved survival times without affecting tension development or morphology. ASM-3T3 microtissues contracted similarly to ex vivo ASM, exhibiting reproducible responses to a range of contractile and relaxant agents. Compared with 2D cultures, microtissues demonstrated identical responses to acetylcholine and KCl, but not histamine, forskolin, or cytochalasin D, suggesting that contractility is regulated by substrate geometry. Microtissues represent a novel model for studying ASM, incorporating a physiological 3D structure, realistic mechanical environment, coculture of multiple cells types, and comparable contractile properties to existing models. This new model allows for rapid screening of biochemical and mechanical factors to provide insight into ASM dysfunction in asthma.

Optimization of human nasal epithelium primary culture conditions for optimal proton oligopeptide and organic cation transporters expression in vitro.

AIM: To investigate the effect of key tissue culture conditions on cell growth, gene expression and functional uptake of peptide and organic cation transporter substrates in the human nasal epithelium (HNE). METHODS: HNE were cultured on different growth surfaces (polystyrene plastic, collagen film, and hydrated collagen gel) and were maintained with three popular nasal tissue culture media supplements [DMEM/F12 supplemented with Ultroser(®) G (2%), FBS (10%) and NuSerum(®) (10%)], respectively. The expression of gene transcripts for organic cation and peptide transporters were screened using qPCR and substrate uptake studies. RESULTS: Cell growth surface (polystyrene plastic surface, dried collagen film and hydrated collagen gel) did not significantly alter gene expression levels. However, Ultroser(®) G and FBS caused significant increase in PEPT1, PEPT2, PHT1, OCT3, and OCTN1 levels (~/=2-5-fold for FBS and 2-8-fold for Ultroser(®) G). In terms of the degree to which the supplements affected gene expression, the following observations were made: effect on OCTN1>PEPT2>OCT3>PHT1>PEPT1. Functional uptake of organic cation (4-Di-1-ASP) and peptide [ß-Ala-Lys (AMCA)] transporter substrates was significantly lower in cells cultured with NuSerum(®) compared to Ultroser(®) G and FBS cultured cells (p>0.05). CONCLUSIONS: Tissue culture media had a major effect on SLC gene expression levels of the human nasal epithelium in primary culture. Ultroser(®) G was identified as the most efficient culture supplement in maintaining SLC transporter expression under most culture conditions, whereas FBS appears to be an economical choice. We do not recommend the use of NuSerum(®) as a supplement for growing HNE for transport studies involving SLC transporters.