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Biomedical subjects

R C Boucher

Publications and source records attributed to R C Boucher.

At least 73 records · Page 4Linked to original sources
PubMed1998

Toxin gene-mediated growth inhibition of lung adenocarcinoma in an animal model of pleural malignancy.

Transduction of malignant cells with toxin genes provides a novel strategy by which to promote tumor cell destruction. Whereas the capacity of the toxin gene/prodrug combination cytosine deaminase/fluorocytosine to inhibit growth of human metastatic pulmonary adenocarcinoma cell lines in vitro is established, the in vivo efficacy of this binary system has not yet been determined. For the development of toxin gene therapy for the treatment of lung adenocarcinoma metastatic to the pleural space, a reliable, disease-specific model is required. The serosa of the rat small intestine resembles the basal lamina of the pleura and provides the basis for a more convenient model than direct injection of tumor into the pleural space. Adenocarcinoma cells are inoculated into everted denuded rat intestine configured as a sac. Immunocytochemical and histological analyses show rapid cell growth with characteristics that mimic nodular metastatic intrapleural disease. In the context of this model, systemically delivered fluorocytosine significantly inhibits the growth of cytosine deaminase-expressing human lung adenocarcinoma cells. The dosing schedule required 30 days; neither addition of an enzyme inhibitor that increases the half-life of fluorocytosine nor intralumenal drug delivery is effective in shortening (to 15 days) the protocol. We conclude that CD continues to hold promise as a toxin gene for lung adenocarcinoma gene therapy, and that prolonged prodrug administration may be required for maximum efficacy.

Adenocarcinoma↗
PubMed1998

A double-blind, placebo controlled, dose ranging study to evaluate the safety and biological efficacy of the lipid-DNA complex GR213487B in the nasal epithelium of adult patients with cystic fibrosis.

UNLABELLED: GTAB1001: A Double-Blind, Placebo Controlled, Dose Ranging Study to Evaluate the Safety and Biological Efficacy of the Lipid-DNA Complex GR213487B in the Nasal Epithelium of Adult Patients with Cystic Fibrosis. OBJECTIVES: To evaluate the effectiveness of various dosages of the lipid-DNA complex GR213487B (0.4375mg and either 4.0mg or 0.0625mg) for producing CFTR gene transfer and correcting the chloride ion transport defect in the nasal epithelium of patients with cystic fibrosis. To assess the safety and tolerability of the lipid-DNA complex GR213487B when applied to the nasal epithelium of patients with cystic fibrosis. DESIGN: Single-center, double-blind, placebo controlled, dose ranging study. DURATION: Pre-treatment evaluations will be performed during two outpatient study visits (ie. between Day -7 to -3 and at Day -2). Patients will be admitted to the Clinical Research Unit (CRU) at the University of North Carolina at Chapel Hill on Day -1 for additional pre-treatment evaluations performed the day prior to administration of double-blind treatment (ie. gene transfer) on Treatment Day 0. Patients will remain in the CRU for 7 days (Day -1 to Day 6) and will be discharged on Day 6. Patients will subsequently be followed on an outpatient basis but will return for another assessment between Days 9-11, and may also return to the CRU for two optional study visits on Days 14 and 21. All patients will return to the CRU on an out-patient basis for follow-up evaluations on Day 28 +/- 3. SETTING: Patients will receive in-patient treatment in the CRU at the University of North Carolina at Chapel Hill and will remain in the CRU for 7 days. PATIENTS: A target enrollment of 12 evaluable patients is planned. STUDY TREATMENTS: Patients who meet all entry criteria will complete pre-treatment assessments, which will take place between Day -7 to Day -1, and will serve as a baseline for specific evaluations and to ensure clinical stability. Patients will return on Day -1 for admission to the CRU the day prior to gene transfer. Each nostril of the patients will be randomly assigned in a double blind manner to receive either GR213487B liquid nasal spray or the lipid alone (ie. control administered as liposome), by topical application directed at the inferior turbinate. The first four patients will receive an initial dosage of GR213487B containing 0.4375 mg of DNA. The decision to proceed to administer a higher dose (ie. 4.0mg DNA) or a lower dose (ie. 0.0625mg DNA) in the subsequent eight patients will be determined by the Principal Investigator in association with an FDA officer serving as an independent Clinical Ombudsman, according to the study plan (see Section 5.5 and Appendix 3-Dosing Flow Chart). MEASUREMENTS: Efficacy Evaluations The primary variables to determine the efficacy of transgene expression will be: * Evidence of vector derived CFTR (cystic fibrosis transmembrane conductance regulator) mRNA, as measured by reverse transcriptase polymerase chain reaction (RT-PCR) in nasal epithelial cells obtained from nasal scrapes on Day 3 and, nasal biopsies on Day 5, if sufficient tissue is available. * Correction of chloride ion transport across the nasal epithelium as measured by the transepithelial electrical potential difference (TEPD). The baseline TEPD will initially be measured, and again subsequently following perfusion of: --zero chloride perfusion containing amiloride (to induce chloride secretion) --zero chloride perfusion containing amiloride and isoproterenol (to increase cAMP-mediated chloride secretion) Secondary measures to determine the efficacy of gene transfer will be: * Evidence of delivery of plasmid DNA in the nasal lavage (Day 1-5, Day 9-11 and Day 28) * Evidence of vector derived CFTR mRNA from nasal scrapes performed after the nasal biopsy (ie. Day 9-11 and/or Day 28) * Percentage of cells from nasal biopsies expressing vector derived CFTR mRNA as measured by in situ hybridization * Evidence of vector derived CFTR

Adult↗
PubMed1998

Effect of host modification and age on airway epithelial gene transfer mediated by a murine leukemia virus-derived vector.

To study retroviral gene transfer to airway epithelia, we used a transient transfection technique to generate high titers (approximately 10(9) infectious units/ml after concentration) of murine leukemia virus (MuLV)-derived vectors pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G). Transformed (CFT1) and primary airway epithelial cells were efficiently transduced by a VSV-G-pseudotyped lacZ vector (HIT-LZ) in vitro. CFT1 cells and primary cystic fibrosis (CF) airway cell monolayers infected with a vector (HIT-LCFSN) containing human CF transmembrane conductance regulator (CFTR) in the absence of selection expressed CFTR, as assessed by Western blot analysis, and exhibited functional correction of CFTR-mediated Cl- secretion. In vitro studies of persistence suggested that pseudotransduction was not a significant problem with our vector preparations. In a sulfur dioxide (SO2) inhalational injury model, bromodeoxyuridine (BrdU) incorporation rates were measured and found to exceed 50% in SO2-injured murine tracheal epithelium. HIT-LZ vector (multiplicity of infection of approximately 10) instilled into the SO2-injured tracheas of anesthetized mice transduced 6.1% +/- 1.3% of superficial airway cells in tracheas of weanling mice (3 to 4 weeks old; n = 10), compared to 1.4 +/- 0.9% in mice 5 weeks of age (n = 4) and 0.2% in mice older than 6 weeks (n = 15). No evidence for gene transfer following delivery of HIT-LZ to tracheas of either weanling or older mice not injured with SO2 was detected. Because only a small fraction of BrdU-labeled airway cells were transduced, we examined the stability of the vector. No significant loss of vector infectivity over intervals (2 h) paralleling those of in vivo protocols was detected in in vitro assays using CFT1 cells. In summary, high-titer vectors permitted complementation of defective CFTR-mediated Cl- transport in CF airway cells in vitro without selection and demonstrated that the age of the animal appeared to be a major factor affecting in vivo retroviral transduction efficiency.

Age Factors↗
PubMed1998

Factors influencing adeno-associated virus-mediated gene transfer to human cystic fibrosis airway epithelial cells: comparison with adenovirus vectors.

Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZ gene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should impact successful gene delivery in CF patients.

Adenoviruses, Human↗
PubMed1998

Limited entry of adenovirus vectors into well-differentiated airway epithelium is responsible for inefficient gene transfer.

Investigations of the efficiency and safety of human adenovirus vector (AdV)-mediated gene transfer in the airways of patients with cystic fibrosis (CF) in vivo have demonstrated little success in correcting the CF bioelectrical functional defect, reflecting the inefficiency of AdV-mediated gene transfer to the epithelial cells that line the airway luminal surface. In this study, we demonstrate that low AdV-mediated gene transfer efficiency to well-differentiated (WD) cultured airway epithelial cells is due to three distinct steps in the apical membrane of the airway epithelial cells: (i) the absence of specific adenovirus fiber-knob protein attachment receptors; (ii) the absence of alphavbeta3/5 integrins, reported to partially mediate the internalization of AdV into the cell cytoplasm; and (iii) the low rate of apical plasma membrane uptake pathways of WD airway epithelial cells. Attempts to increase gene transfer efficiency by increasing nonspecific attachment of AdV were unsuccessful, reflecting the inability of the attached vector to enter (penetrate) WD cells via nonspecific entry paths. Strategies to improve the efficiency of AdV for the treatment of CF lung disease will require methods to increase the attachment of AdV to and promote its internalization into the WD respiratory epithelium.

Adenoviridae↗
PubMed1998

Effect of in vivo corticosteroids on Na+ transport across airway epithelia.

We have investigated the role in vivo of mineralocorticoid and glucocorticoid hormones in regulating the rate of electrogenic amiloride-sensitive Na+ absorption across murine airway tissue studied in vivo (nasal potential difference) and in vitro (Ussing chambers). We found that elevating the plasma aldosterone concentration 10-fold (low-Na+ diet) had no significant effect on amiloride-sensitive Na+ absorption across tracheal or nasal epithelia. High doses of dexamethasone for 1 wk likewise did not change the rate of amiloride-sensitive Na+ absorption across airway epithelia. In contrast, both hormonal manipulations elevated the rate of colonic Na+ absorption. Furthermore, adrenalectomy (both normal and cystic fibrosis mice) also failed to alter Na+ absorption across airway epithelia. We conclude that, in vivo, neither the mineralocorticoid nor the glucocorticoid hormones significantly regulate the rates of amiloride-sensitive electrogenic Na+ absorption across airway epithelia in the adult mouse.

Adrenal Cortex Hormones↗
PubMed1998

Effects of topically delivered benzamil and amiloride on nasal potential difference in cystic fibrosis.

The raised nasal transepithelial potential difference (PD) in cystic fibrosis (CF) reflects accelerated active transport of Na+, and is inhibited by topical administration of the Na+ channel blocker, amiloride. The aim of this study was to investigate the dose-effect and time course of topically administered Na+ conductance inhibitors to inhibit nasal PD, including benzamil, an analog of amiloride. We measured the magnitude of drug inhibition of Na+ transport [percent inhibition of baseline PD (DeltaPD%)] and duration of inhibition of PD, defined as the time when drug inhibition of PD had recovered by 50% (effective time = ET50). Amiloride [10(-)3 M (n = 16), 3 x 10(-)3 M (n = 9), 6 x 10(-)3 M (n = 7), 10(-)2 M (n = 3)] or benzamil [1.7 x 10(-)3 M (n = 7), and 7 x 10(-)3 M (n = 5)] were administered to the nasal surface via an aerosol generated by a jet nebulizer and a nasal mask. The concentration-dependent magnitude (DeltaPD%) of inhibition was similar for amiloride and benzamil ( approximately 67- 77%), whereas the duration of inhibition (ET50) was about two-and-a-half times longer after benzamil administration as compared with equivalent concentrations of amiloride [1.6 +/- 0. 06 versus 4.5 +/- 0.6 h (ET50 +/- SEM), at 6-7 x 10(-)3 M]. In vitro studies of cultured normal nasal epithelia demonstrated directly that benzamil induced an approximately 2-fold more prolonged inhibition of active Na+ transport than amiloride. These data suggest aerosolized benzamil is a candidate long-duration Na+ channel blocker for CF.

Administration, Intranasal↗
PubMed1998

Transduction of non-small cell lung cancer cells by adenoviral and retroviral vectors.

Gene transfer into a panel of non-small cell lung cancer (NSCLC) cells by adenoviral (Ad) and retroviral (RV) vectors was studied. Indexed to multiplicity of infection (MOI), Ad vectors transduce squamous, adenosquamous, and malignant mesothelioma cells with greater efficiency than large cells or adenocarcinoma cells. Transduction-sensitive cells bind the Ad vector with specificity for the Ad fiber knob, and internalize vector efficiently. Transduction-refractory cells bind and internalize vector by less efficient pathways. Like Ad vectors, there is heterogeneity in RV transduction efficiencies of different NSCLC subtypes. With respect to the most common cell type metastatic to the pleural space (adenocarcinoma), amphotropic retroviral vectors transduce cells of this subtype more efficiently (at a lower MOI) than Ad. RV transduction is not solely dependent on cellular replication, and both permissive and refractory cell lines express the mRNA for the amphotropic RV receptor. These observations suggest that neither Ad nor RV vectors will suffice a priori as the optimal gene transfer vehicle, and successful gene therapy of lung cancer may require tumor-specific or patient-specific vectors.

Adenoviridae↗
PubMed1998

Effects of ion composition and tonicity on human neutrophil antibacterial activity.

Infants with cystic fibrosis (CF) often are infected with Staphylococcus aureus (S. aur.), which is followed by colonization with Pseudomonas aeruginosa (P. aerug.). In spite of an excessive, neutrophil-dominated inflammatory response in the respiratory tract, patients with CF often succumb to pulmonary infections with P. aerug. Because peripheral blood neutrophils of these patients have normal functions, we examined whether hypothesized alterations of the airway surface liquids (ASL) in these patients significantly impair neutrophil bactericidal activity in the microenvironment of the CF lung. The ionic composition of CF ASL is still not entirely defined and has been speculated to be abnormally high or abnormally low in Na+ and Cl- concentrations; estimates of osmolarities have ranged from 200 (hypo-osmolar) to 285 (iso-osmolar) to > 300 meq/L (hyper-osmolar). Our data indicate that bacterial killing activity of human peripheral blood neutrophils against P. aerug. or S. aur. is not decreased in buffers in which NaCl was replaced with equimolar concentrations of choline Cl, KCl, or N-methyl-D-glucamine chloride to maintain isotonicity. Amiloride or benzamil, known modulators of Na+ transport in neutrophils, did not interfere with this neutrophil function. Deviations from isotonicity of +/- 50% also failed to diminish bactericidal activity of neutrophils significantly. In contrast, superoxide production and enzyme secretion in response to the chemotactic peptide N-formylmethionylleucylphenylalanine appeared to be sensitive to the ionic milieu of the assay buffers. Our results suggest that the postulated alterations in the ionic composition of ASL in CF lungs are insufficient to explain why neutrophils fail to clear infections with P. aerug. in these patients.

Adult↗
PubMed1997

Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects.

To test whether a major contribution of airways epithelial ion transport to lung defense reflects the regulation of airway surface liquid (ASL) ionic composition, we measured ASL composition using the filter paper technique. On nasal surfaces, the Cl- concentration (approximately 125 meq/liter) was similar to plasma, but the Na+ concentration (approximately 110 meq/liter) was below plasma, and K+ concentration (approximately 30 meq/liter) above plasma. The resting ASL osmolarity [2(Na+ + K+); 277 meq/liter] approximated isotonicity. There were no detectable differences between cystic fibrosis (CF) and normal subjects. In the lower airways, the Na+ concentrations were 80-85 meq/liter, K+ levels approximately 15 meq/liter, and Cl- concentrations 75-80 meq/liter. Measurements of Na+ activity with Na(+)-selective electrodes and osmolality with freezing point depression yielded values consistent with the monovalent cation measurements. Like the nasal surfaces, no differences in cations were detected between CF, normal, or chronic bronchitis subjects. The tracheobronchial ASL hypotonicity was hypothesized to reflect collection-induced gland secretion, a speculation consistent with observations in which induction of nasal gland secretion produced hypotonic secretions. We conclude that there are no significant differences in ASL ion concentrations between CF, normal, and chronic bronchitis subjects and, because ASL ion concentrations exceed values consistent with defensin activity, the failure of CF lung defense may reflect predominantly factors other than salt-dependent defensins.

Adult↗
PubMed1997

Direct demonstration of mechanically induced release of cellular UTP and its implication for uridine nucleotide receptor activation.

ATP is released from most cell types and functions as an extracellular signaling molecule through activation of members of the two large families of P2X and P2Y receptors. Although three mammalian P2Y receptors have been cloned that are selectively activated by uridine nucleotides, direct demonstration of the release of cellular UTP has not been reported. Pharmacological studies of the P2Y4 receptor expressed in 1321N1 human astrocytoma cells indicated that this receptor is activated by UTP but not by ATP. Mechanical stimulation of 1321N1 cells also resulted in release of a molecule that markedly activated the expressed P2Y4 receptor. This nucleotide was shown to be UTP by two means. First, high performance liquid chromatography analysis of the medium from [33P]H3PO4-loaded 1321N1 cells illustrated that mechanical stimulation resulted in a large increase in a radioactive species that co-eluted with authentic UTP. This species was degraded by incubation with the nonspecific pyrophosphohydrolase apyrase or with hexokinase and was specifically lost by incubation with the UTP-specific enzyme UDP-glucose pyrophosphorylase. Second, a sensitive assay that quantitates UTP mass at low nanomolar concentrations was devised based on the nucleotide specificity of UDP-glucose pyrophosphorylase. Using this assay, mechanical stimulation of 1321N1 cells was shown to result in an increase of medium UTP levels from 2.6 to 36.4 pmol/10(6) cells within 2 min. This increase was paralleled by a similar augmentation of extracellular ATP levels. A calcein-based fluorescence quenching method was utilized to confirm that none of the increases in medium nucleotide levels could be accounted for by cell lysis. Taken together, these results directly demonstrate the mechanically induced release of UTP and illustrate the efficient coupling of this release to activation of P2Y4 receptors.

Adenosine Triphosphate↗
PubMed1997

Identification of an ecto-nucleoside diphosphokinase and its contribution to interconversion of P2 receptor agonists.

The P2Y4 receptor is selectively activated by UTP. Although addition of neither ATP nor UDP alone increased intracellular Ca2+ in 1321N1 human astrocytoma cells stably expressing the P2Y4 receptor, combined addition of these nucleotides resulted in a slowly occurring elevation of Ca2+. The possibility that the stimulatory effect of the combined nucleotides reflected formation of UTP by an extracellular transphosphorylating activity was investigated. Incubation of cells with [3H]UDP or [3H]ADP under conditions in which cellular release of ATP occurred or in the presence of added ATP resulted in rapid formation of the corresponding triphosphates. Transfer of the gamma-phosphate from [gamma-33P]ATP to nucleoside diphosphates confirmed that the extracellular enzymatic activity was contributed by a nucleoside diphosphokinase. The majority of this activity was associated with the cell surface of 1321N1 cells, suggesting involvement of an ectoenzyme. Both ADP and UDP were effective substrates for transphosphorylation. Since ecto-nucleotidase(s) has been considered previously to be the primary enzyme(s) responsible for metabolism of extracellular nucleotides, the relative rates of hydrolysis of ATP, ADP, UTP, and UDP also were determined for 1321N1 cells. All four nucleotides were hydrolyzed with similar Km and Vmax values. Kinetic analyses of the ecto-nucleoside diphosphokinase and ecto-nucleotidase activities indicated that the rate of extracellular transphosphorylation exceeds that of nucleotide hydrolysis by up to 20-fold. Demonstration of the existence of a very active ecto-nucleoside diphosphokinase together with previous observations that stress-induced release of ATP occurs from most cell types indicates that transphosphorylation is physiologically important in the extracellular metabolism of adenine and uridine nucleotides. Since the P2Y receptor class of signaling proteins differs remarkably in their respective specificity for adenine and uridine nucleotides and di- and triphosphates, these results suggest that extracellular interconversion of adenine and uridine nucleotides plays a key role in defining activities in nucleotide-mediated signaling.

Adenosine Diphosphate↗
PubMed1997

Cystic fibrosis transmembrane conductance regulator inverts protein kinase A-mediated regulation of epithelial sodium channel single channel kinetics.

Abnormal regulation of ion channels by members of the ABC transport protein superfamily has been implicated in hyperinsulinemic hypoglycemia and in excessive Na+ absorption by airway epithelia in cystic fibrosis (CF). How ABC proteins regulate ion conductances is unknown, but must generally involve either the number or activity of specific ion channels. Here we report that the cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in CF, reverses the regulation of the activity of single epithelial sodium channels (ENaC) by cAMP. ENaC expressed alone in fibroblasts responded to activation of cAMP-dependent protein kinase with increased open probability (Po) and mean open time, whereas ENaC co-expressed with CFTR exhibited decreased Po and mean open time under conditions optimal for PKA-mediated protein phosphorylation. Thus, CFTR regulates ENaC at the level of single channel gating, by switching the response of single channel Po to cAMP from an increase to a decrease.

3T3 Cells↗
PubMed1997

Retroviral gene transfer is inhibited by chondroitin sulfate proteoglycans/glycosaminoglycans in malignant pleural effusions.

Gene therapy may be an important adjuvant for treating cancer in the pleural space. The initial results of retroviral gene transfer to cancer cells in malignant pleural effusions revealed that transduction was markedly inhibited, and studies to characterize the inhibitory factor(s) were performed. The inhibition was contained within the soluble, rather than cellular, components of the effusions and was demonstrated with amphotropic, gibbon ape leukemia virus, and vesicular stomatitis virus-glycoprotein pseudotyped retroviral vectors. After excluding complement proteins, a series of studies identified chondroitin sulfates (CSs) as the inhibitory substances. First, treatment of the effusions with mammalian hyaluronidase or chondroitinases, but not Streptomyces hyaluronidase, abolished the inhibitory activity. Second, addition of exogenous CS glycosaminoglycans mimicked the inhibition observed with pleural effusions. Third, immunoassays and biochemical analyses of malignant pleural effusion specimens revealed CS in relevant concentrations within pleural fluid. Fourth, proteoglycans/glycosaminoglycans isolated from the effusions inhibited retroviral gene transfer. Analyses of the mechanism of inhibition indicate that the chondroitin sulfates interact with vector in solution rather than at the target cell surface. These results suggest that drainage of the malignant pleural effusion, and perhaps enzymatic pretreatment of the pleural cavity, will be necessary for efficient retroviral vector mediated gene delivery to pleural metastases.

Animals↗
PubMed1997

UDP activates a mucosal-restricted receptor on human nasal epithelial cells that is distinct from the P2Y2 receptor.

The presence of the P2Y2 (P(2U)-purinergic) receptor on the apical surface of airway tissue raises the possibility that aerosolized UTP might be used therapeutically to induce Cl- secretion in individuals with cystic fibrosis. However, the duration of the effects of UTP may be limited by enzymatic degradation. We therefore have analyzed the metabolism of UTP and its metabolite UDP on polarized human nasal epithelium (HNE), and have compared the pharmacological activities of these two uridine nucleotides. HPLC analysis of medium bathing the mucosal surface of HNE cells revealed the presence of an ecto-nucleotidase(s) that hydrolyzed [3H]UTP and [3H]UDP with t1/2 values (at 1 microM nucleotide) of 14 and 27 min, respectively. An ecto-nucleoside diphosphokinase activity also was observed, which promoted conversion of [3H]UDP into [3H]UTP in the presence of ATP. The effects of UDP on [3H]inositol phosphate accumulation, intracellular calcium levels ([Ca2+]i), and Cl- secretory rates (I(Cl-)) were quantitated in HNE cells in the presence of hexokinase and glucose to ensure that no UTP (or ATP) contaminated UDP solutions during the assays. Although UDP does not activate the human P2Y2 receptor, mucosal addition of UDP promoted [3H]inositol phosphate accumulation with an EC50 of 190 nM. Mucosal addition of UTP stimulated [3H]inositol phosphate accumulation with an EC50 of 280 nM. The maximal effects of mucosal UDP on [3H]inositol phosphate, [Ca2+]i, and I(Cl-) responses were approximately one-half of those observed with mucosal UTP. Serosal application of UTP promoted a 50% greater [3H]inositol phosphate and calcium response than did mucosal application of UTP. In contrast, UDP had no effect when added to the serosal medium. Repetitive mucosal applications of UDP to HNE cells resulted in a progressive loss, i.e., desensitization, of the [Ca2+]i and I(Cl-) response to UDP, whereas the corresponding responses to UTP remained unchanged. Our results provide evidence for the existence of a UDP receptor on HNE cells that is pharmacologically distinct from the P2Y2 receptor. The relative stability of UDP on the airway surface and the apparent predominant mucosal expression of this putative UDP receptor make it a potential target for cystic fibrosis treatment.

Calcium↗
PubMed1997

CFTR-like chloride channels in non-ciliated bronchiolar epithelial (Clara) cells.

Distal airways are believed to be a major site of disease in cystic fibrosis. The product of the CF gene, CFTR, is expressed in non-ciliated bronchiolar epithelial (Clara) cells. Clara cells in primary culture are capable of Cl- secretion which can be stimulated by cAMP and extracellular nucleotides. We used the patch clamp technique to look for Cl- channels in the apical membrane of rabbit Clara cells. In cell-attached patches, we recorded Cl- channels with a conductance for outward currents of 7.5 +/- 0.4 pS (n = 10) and for inward currents of 3.2 +/- 0.5 pS (n = 10); these channels typically exhibited slow kinetics and were not inhibited by the Cl- channel blockers NPPB and DIDS. Channel activity was not noticeably dependent on pipette potential. Addition of chlorophenylthio-cyclic AMP (cpt-cAMP) to the bath increased the percentage of cell-attached patches with active channels (33.8% vs 56.7%; p< 0.05) and the channel open probability (0.49 +/- 0.03 vs 0.84 +/- 0.02; p< 0.05). Extracellular ATP increased the percentage of cell-attached patches with active channels (28.7% to 50.0%; p< 0.05) but had no significant effect on the channel open probability (0.62 +/- 0.07 vs 0.60 +/- 0.06). In conclusion, rabbit non-ciliated bronchiolar epithelial cells express low-conductance Cl- channels that share many similarities with the CFTR-related Cl- channel and are regulated by cAMP and extracellular ATP.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

Loss of binding and entry of liposome-DNA complexes decreases transfection efficiency in differentiated airway epithelial cells.

The target cells for gene therapy of cystic fibrosis lung disease are the well differentiated cells that line airway lumens. Employing cultures of airway epithelial cells that grow like "islands" and exhibit a continuum of cellular differentiation, we studied the mechanisms that render well differentiated cells more difficult to transfect with cationic liposomes than poorly differentiated cells. The poorly differentiated cells at the edge of the islands were transfectable with liposome-DNA complexes (pCMVbeta:LipofectACE = 1:5 (w/w)), whereas the more differentiated cells in the center of the islands were not. Evaluation of the steps leading to lipid-mediated transfection revealed that edge cells bound more liposome-DNA complexes, in part due to a more negative surface charge (as measured by cationized ferritin binding), and that edge cells internalized more liposome-DNA complexes than central cells. Edge cells exhibited receptor-mediated endocytosis of LDL, pinocytosis of 10-nm microspheres, and phagocytosis of 2-microm microspheres, whereas central cells were only capable of receptor-mediated endocytosis. Cytochalasin B, which inhibited pinocytosis by 65% and phagocytosis by 93%, decreased edge cell liposome-DNA complex entry by 50%. Potassium depletion, which decreased phagocytosis by >90% but had no effect on pinocytosis, inhibited edge cell liposome-DNA complex entry by 71%. These results indicate that liposome-DNA complexes enter edge cells via phagocytosis and that this pathway is not detectable in central cells. In conclusion, both reduced negative surface charge and absence of phagocytosis internalization pathways in relatively differentiated cells may explain differentiation-dependent decrements in cationic liposome-mediated gene transfer in airway epithelia.

Animals↗