Human Nasal Cell Research Articles

In situ-gelling hydrophobized starch nanoparticle-based nanoparticle network hydrogels for the effective delivery of intranasal olanzapine to treat brain disorders

Intranasal (IN) delivery offers potential to deliver antipsychotic drugs with improved efficacy to the brain. However, the solubilization of such drugs and the frequency of required re-application both represent challenges to its practical implementation in treating various mental illnesses including schizophrenia. Herein, we report a sprayable nanoparticle network hydrogel (NNH) consisting of hydrophobically-modified starch nanoparticles (SNPs) and mucoadhesive chitosan oligosaccharide lactate (COL) that can gel in situ within the nasal cavity and release ultra-small penetrative SNPs over time. Hydrophobization of the SNPs enables enhanced uptake and prolonged release of poorly water soluble drugs such as olanzapine from the NNH depot through mucous and ultimately into the brain via the nose-to-brain (N2B) pathway. The hydrogel shows high in vitro cytocompatibility in mouse striatal neuron and human primary nasal cell lines and in vivo efficacy in an amphetamine-induced pre-clinical rat schizophrenia model, with IN-delivered NNH hydrogels maintaining successful attenuation of locomotor activity for up to 4 h while all other tested treatments (drug-only IN or conventional intraperitoneal delivery) failed to attenuate at any time point past 0.5 h. As such, in situ-gelling NNHs represent a safe excipient for the IN delivery of hydrophobic drugs directly to the brain using customized SNPs that exhibit high penetration and drug complexing properties to maximize effective drug delivery.

Gold Nanoparticles: Tunable Characteristics and Potential for Nasal Drug Delivery.

A general procedure to prepare gold nanourchins (GNUs) via a seed-mediated method was followed using dopamine hydrochloride as a reducing agent and silver nitrate salt (AgNO3) as a shape-directing agent. The novelty of this study comes from the successful incorporation of the prepared gold urchins as an aqueous suspension in a nasal pressurized metered dose inhaler (pMDI) formulation and the investigation of their potential for olfactory targeting for direct nose-to-brain drug delivery (NTBDD). The developed pMDI formulation was composed of 0.025% w/w GNUs, 2% w/w Milli-Q water, and 2% w/w EtOH, with the balance of the formulation being HFA134a propellant. Particle integrity and aerosolization performance were examined using an aerosol exposure system, whereas the nasal deposition profile was tested in a sectioned anatomical replica of human nasal airways. The compatibility of the gold dispersion with the nasal epithelial cell line RPMI 2650 was also investigated in this study. Colloidal gold was found to be stable following six-month storage at 4 °C and during the lyophilization process utilizing a pectin matrix for complete re-dispersibility in water. The GNUs were intact and discrete following atomization via a pMDI, and 13% of the delivered particles were detected beyond the nasal valve, the narrowest region in the nasal cavity, out of which 5.6% was recovered from the olfactory region. Moreover, the formulation was found to be compatible with the human nasal epithelium cell line RPMI 2650 and excellent cell viability was observed. The formulated GNU-HFA-based pMDI is a promising approach for intranasal drug delivery, including deposition in the olfactory region, which could be employed for NTBDD applications.

Investigation of the inhibitory effects of simvastatin in RPMI 2650: An in-vitro study

Objective: The present study was carried out to investigate the antiproliferative effects of simvastatin involving Human Squamous Nasal Cell Carcinoma (RPMI 2650 cell line), which is one of the most common head and neck cancers with the highest incidence and mortality rates in Asian countries. The anti-cancer effects of simvastatin in NPC have not yet been investigated in depth, hence it will be illustrated in the present study. Materials and methods: The cells were treated with various concentrations of Simvastatin in a dose dependent (0, 0.1, 0. 4, 3.0 mg/ml) and time dependent (24, 48 and 72 hours) manner. The cancer cell viability was then assessed by using MTT assay at absorbance of 590 nm. Results: Simvastatin induced reduction in cell viability number and induced apoptosis in RPMI 2650 cell line. Simvastatin for 72 h significantly reduced cell growth as compared to 48 and 24 h pre-incubation with simvastatin treatment. After 72 h incubation with 0.1 mg/ml, 0.4 mg/ml and 3.0 mg/ml simvastatin, the cell viability decreased from 100% in treated control cells to 23%, 21% and 14% respectively. Conclusions: This finding demonstrate the potential of simvastatin and probably may have therapeutic benefit for NPC cell growth.

IN VITRO CYTOTOXIC EVALUATION AND APOPTOTIC EFFECTS OF DILLAPIOLE ON HUMAN NASAL SQUAMOUS CELL CARCINOMA

Dillapiole is one of the major compounds in Peperomia pellucida. Dillapiole exerts cytotoxicity effect on several cancer cells such as breast and colon with a minimal effect on normal cells. However, its effect on cancers in head and neck region remains ambiguous. Therefore, in the current study, the cytotoxicity effect of dillapiole on human nasal squamous cell carcinoma, RPMI 2650 and the underlying mechanism were investigated. Normal human gingival fibroblast, HGnF cells was used as comparison. Cisplatin and untreated cells were used as positive and negative controls, respectively. Cell cytotoxicity was determined using WST-1 assay; and validated by trypan blue exclusion assays. Cell death mechanism was determined using Annexin V-FITC detection kit and analyzed by flow cytometer. The cytotoxic effect of dillapiole on RPMI 2650 cells was shown significantly increased with the increasing amount of dillapiole from 25.93 ± 6.39 % to 83.87 ± 8.43 % (p<0.05, n=3) with the respective IC50 and IC75 of 46 µM and 125 µM. None of both IC50 and IC75 were obtained for HGnF cells up to 150 µM. Loss of normal shape, cytoplasm shrinkage and reduction in cell volume were detected in RPMI 2650 cells after dillapiole treatment at respective IC50 (46 µM) and IC75 (125 µM), indicating apoptosis. These findings indicate that dillapiole cytotoxicity effect is more selective towards RPMI 2650 compared to HGnF cells and the mechanism of death was through the induction of apoptosis.

Temporary Storage of the Human Nasal Tissue and Cell Sheet for Wound Repair.

Temporary storage of nasal tissues and nasal cell sheets, which entails transportation between hospitals and cell culture facilities, is an important issue in regenerative medicine. Herein, we investigated the preservation of chilled and frozen nasal tissues and expiry dates of ready-to-use nasal cell sheets. Although the cell number in preserved tissues was lower than that in fresh tissue, nasal cell sheets could be fabricated from tissues that had been refrigerated for 5 days and frozen–thawed over 5 days. Moreover, the nasal mucosal cell sheets were preserved in a non-hazardous buffer. The cell number, viability, and structure were not maintained in saline containing E-cadherin for 2 days; however, these were maintained in Hank’s balanced salt solution for 2 days, but not for 5 days. To assess the proliferation capacity of cells in the stored cell sheets, we performed cell sheet grafting assays in vitro. Cell sheets stored in Hank’s balanced salt solution for 2 days adhered to collagen gel and expanded normally. Our results show that nasal tissues can be stored temporarily in refrigerators or deep freezers, and Hank’s balanced salt solution can be used for preservation of ready-to-use cell sheets for a few days.

Cytokines, Proliferation Markers, Antimicrobial Factors and Neuropeptide-Containing Innervation in Human Nasal Mucosa after Rhinoseptoplasty Procedure.

The nasal cavity lined by nasal mucosa, is a significant part of respiratory system of human. However, there are no studies aimed to detect a molecular phenotype of healthy and normal functioning nasal mucosa, obtained after rhinoseptoplasty procedure, to understand its physiology and growth and inflammation processes. Thus, our aim is to identify human healthy nasal mucosa cytokines, neuropeptide-containing innervation and cell proliferation markers to form a control group for further tissue investigation of human nasal polyposis as the next step of our research. The study included surgery materials from 17 healthy humans. Biotin-streptavidin immunohistochemistry was performed for detection of tissue PGP9.5, Ki-67, β-Defensin 2, IL-1, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12. Results were evaluated semi-quantitatively and by Friedman ANOVA and Spearman rang correlation tests. All factors were more widely expressed by superficial epithelium than by glandular one. Abundance of ILs-8, -10 and -12 positive cells was detected in comparison with moderate to numerous distributions of IL-1, IL-6 and β-Defensin 2. Moderate number of PGP 9.5-containing nerve fibers and only few to moderate Ki-67 positive cells were found in healthy nasal mucosa. We revealed statistically significant difference between Ki-67 and ILs-4, -6, -7, -8, -10, -12 both in healthy nasal mucosa superficial and glandular epithelium. From nasal epithelia, commonly the surface one displays more cytokines and β-Defensin 2 in comparison to the glandular one. Numerous to abundant expression of ILs-4, -6, -7, -8, -10, -12 and β-Defensin 2 in nasal superficial and glandular epithelia proves probably these factors’ role into the common immune response of tissue and stimulation of immune cell differentiation.

On the in vivo origin of human nasal mesenchymal stem cell cultures.

ObjectivesMesenchymal stem cells (MSCs), classically expanded in culture from bone marrow, are of broad interest to the regenerative medicine community. Human nasal turbinate mesenchymal‐like stem cell cultures have also been described, defined by an in vitro phenotype similar to bone marrow MSCs. Nonetheless, the identity in vivo of the cells that give rise to nasal MSC‐like cultures remains unclear, and these cells are often suggested to be related to olfactory lineages. Here, we sought to define the in vivo phenotype of human nasal MSC‐like cells.MethodsHuman turbinate tissue samples were used for RNA and immunohistochemical analysis. We also analyzed a recently published single cell RNA‐sequencing dataset from adult human olfactory and respiratory mucosa samples from our lab, to focus on cell populations expressing MSC markers. Immunochemistry was performed to stain turbinate sections and nasal MSC cultures for selected markers.ResultsWhile there is no single MSC‐specific gene, we identified a human nasal mucosal cell population in vivo that uniquely expressed transcripts characteristic of typical MSC cultures, including ENG (CD105), NES, and CD34, and lacked expression of other transcripts associated with surface epithelia. The expression of transcription factors such as SOX17, EBF1, and FOXP1 suggests cells in the MSC‐like cluster maintain an ability to direct cell fate, consistent with the behavior of nasal MSC‐like cells in vitro. SOX17 was found to be uniformly expressed by nasal MSC cultures, consistent with the in vivo data. Immunohistochemistry of human nasal tissue samples indicated that ENG, CD34, and SOX17 expression localized selectively to cells surrounding blood vessels in the lamina propria.ConclusionOur findings provide evidence that the in vivo origin of nasal MSC‐like cultures is likely a vascular or pericyte population, rather than cells related to the olfactory neuronal lineage.Level of evidenceNA.

Different Culture Conditions Affect Drug Transporter Gene Expression, Ultrastructure, and Permeability of Primary Human Nasal Epithelial Cells.

This study aimed to characterize a commercially available primary human nasal epithelial cell culture and its gene expression of a wide range of drug transporters under different culture conditions. Human nasal cells were cultured in three different types of culture media at the air-liquid (A-L) or liquid-liquid (L-L) interfaces for 1 or 3 wks. The effects of the different cell culture conditions were evaluated using light and electron microscopy, transepithelial electrical resistance (TEER) measurements, permeation studies with dextran, and gene expression profiling of 84 drug transporters. The type of culture medium affected cell ultrastructure, TEER, and dextran permeation across epithelia. The expression of 20 drug transporter genes depended on the culture interface and/or time in culture; the A-L interface and longer time in culture favored higher expression levels of five ABC and seven SLC transporters. Culture conditions influence the morphology, barrier formation, permeation properties, and drug transporter expression of human nasal epithelial cells, and this must be taken into consideration during the establishment and validation of in vitro models. A thorough characterization of a nasal epithelial model and its permeability properties is necessary to obtain an appropriate standardized model for the design of aerosol therapeutics and drug transport studies.

Quantitative analysis of nasal transcripts reveals potential biomarkers for Parkinson\u2019s disease

Patients with Parkinson’s disease (PD) oftentimes develop olfactory dysfunction in their early stages, converting the nasal environment into a useful source of potential biomarkers. Here we determined the possible application of nasal fluid cells for PD biomarker identification. Thirty PD patients and 13 age-matched healthy controls were enrolled in this study. Messenger RNA levels of selected PD-related genes were monitored through real-time quantitative PCR. Target gene transcripts can be efficiently amplified from the cDNA library from human nasal fluid cell pellets. And subsequent analysis showed both a marked downregulation of parkin transcripts and an upregulation of AIMP2 in PD patients when compared to controls (cutoff value = 1.753 for with 84.2% sensitivity and 84.6% specificity; 0.359 for parkin with 76.7% sensitivity and 76.9 specificity). Moreover, alteration pattern of parkin and AIMP2 in PD was distinct from another neurodegenerative disease, multiple system atrophy. Analysis in both the early and late stages of PD cases reported that parkin levels inversely correlated with PD stages. Our results validate the practical value of easily accessible nasal fluid cells and the utility of both AIMP2 and parkin as potential biomarkers for PD diagnosis.

Analysis of human nasal mucosal cell sheets fabricated using transported tissue and blood specimens.

Previously, we succeeded in transplanting autologous nasal mucosal cell sheets in the middle ears of 5 patients, who underwent cholesteatoma resection, which prevents recurrence of cholesteatoma in clinical settings. Current good manufacturing practice (GMP) standards for human cell cultivation requires the establishment of cell processing centers (CPC) which act as germ-free facilities. However, due to practical difficulties involved in establishing and maintaining such facilities at each individual hospital, a functional transport system is felt to be needed for the continuation of effective regenerative therapy. In the current study, nasal mucosal tissue and autologous blood obtained from 3 human volunteers were transported for over 3 h. Disinfected nasal tissues were cultured using keratinocyte culture medium, which included autologous serum prepared from blood. After 24 d, cultured nasal mucosal cells were transported for over 3 h and subsequently assessed for cell number, viability and purity. Moreover, CK4, CK8, and CK18 were analyzed the suitability of these nasal mucosal cell sheets for middle ear regenerative therapy.Overall, we confirmed that nasal mucosal cell sheets can be fabricated using transported nasal mucosal tissue and blood. This study would be contribute to establish a new regenerative therapy for clinical application, accompanied with transportation between companies and hospitals.

Chitosan-Coated Nanoparticles: Effect of Chitosan Molecular Weight on Nasal Transmucosal Delivery.

Drug delivery to the brain represents a challenge, especially in the therapy of central nervous system malignancies. Simvastatin (SVT), as with other statins, has shown potential anticancer properties that are difficult to exploit in the central nervous system (CNS). In the present work the physico–chemical, mucoadhesive, and permeability-enhancing properties of simvastatin-loaded poly-ε-caprolactone nanocapsules coated with chitosan for nose-to-brain administration were investigated. Lipid-core nanocapsules coated with chitosan (LNCchit) of different molecular weight (MW) were prepared by a novel one-pot technique, and characterized for particle size, surface charge, particle number density, morphology, drug encapsulation efficiency, interaction between surface nanocapsules with mucin, drug release, and permeability across two nasal mucosa models. Results show that all formulations presented adequate particle sizes (below 220 nm), positive surface charge, narrow droplet size distribution (PDI < 0.2), and high encapsulation efficiency. Nanocapsules presented controlled drug release and mucoadhesive properties that are dependent on the MW of the coating chitosan. The results of permeation across the RPMI 2650 human nasal cell line evidenced that LNCchit increased the permeation of SVT. In particular, the amount of SVT that permeated after 4 hr for nanocapsules coated with low-MW chitosan, high-MW chitosan, and control SVT was 13.9 ± 0.8 μg, 9.2 ± 1.2 µg, and 1.4 ± 0.2 µg, respectively. These results were confirmed by SVT ex vivo permeation across rabbit nasal mucosa. This study highlighted the suitability of LNCchit as a promising strategy for the administration of simvastatin for a nose-to-brain approach for the therapy of brain tumors.

Retracted: Cytotoxic effects of dillapiole on human nasal squamous cell carcinoma, RPMI 2650 cells

Retracted: Cytotoxic effects of dillapiole on human nasal squamous cell carcinoma, RPMI 2650 cells

Evaluation of antifibrotic effect of pirfenidone on human nasal mucosal fibroblast cell culture

Background: One of the main reasons of failure in surgical treatment of primary acquired nasolacrimal duct obstruction is excessive postoperative scarring of the dacryostomy. Despite the variety of procedures designed to prevent this, conflicting evidence of their efficacy and safety provide incentive for further research of antifibrotic therapeutics for adjunctive use in dacryocystorhinostomy.Aims: To evaluate the antifibrotic effect of pirfenidone on human nasal mucosal fibroblast cell culture.Materials and methods: Human nasal mucosal fibroblast cell cultures were established using samples obtained from 3 consecutive patients undergoing endonasal endoscopic dacryocystorhinostomy. Cell viability following treatment with pirfenidone was evaluated using MTS-assay. Induced inhibition of cell proliferation and migration was determined using scratch wound assay.Results: In this study pirfenidone exhibited a significant dose-dependent inhibiting effect on fibroblast proliferation with insignificant cell toxicity. Cell viability following 48 hours of incubation with various pirfenidone concentrations did not drop below 80%. The recovery of the fibroblast monolayer assessed after 24 hours of incubation was 84.88 and 8.26% in the control group, at a drug concentration of 0.15 mg/ml. Cell proliferation and migration was severely inhibited in cell culture specimens treated with pirfenidone compared to controls. The difference between groups was statistically significant (p=0,001).Conclusions: In our study pirfenidone demonstrated a pronounced antifibrotic effect. It is unlikely that inhibition of proliferation and migration of human nasal mucosal fibroblasts is mediated by cell toxicity of this medication as it was evaluated as low. Nonetheless an in vitro analysis is insufficient to judge pirfenidone’s efficacy and safety in preventing cicatrix formation following dacrycystorhinostomy.

Detection of CFTR function and modulation in primary human nasal cell spheroids

BackgroundExpansion of CFTR modulators to patients with rare/undescribed mutations will be facilitated by patient-derived models quantifying CFTR function and restoration. We aimed to generate a personalized model system of CFTR function and modulation using non-surgically obtained nasal epithelial cells (NECs). MethodsNECs obtained by curettage from healthy volunteers and CF patients were expanded and grown in 3-dimensional culture as spheroids, characterized, and stimulated with cAMP-inducing agents to activate CFTR. Spheroid swelling was quantified as a proxy for CFTR function. ResultsNEC spheroids recapitulated characteristics of pseudostratified respiratory epithelia. When stimulated with forskolin/IBMX, spheroids swelled in the presence of functional CFTR, and shrank in its absence. Spheroid swelling quantified mutant CFTR restoration in F508del homozygous cells using clinically available CFTR modulators. ConclusionsNEC spheroids hold promise for understanding rare CFTR mutations and personalized modulator testing to drive evaluation for CF patients with common, rare or undescribed mutations.Portions of this data have previously been presented in abstract form at the 2016 meetings of the American Thoracic Society and the 2016 North American Cystic Fibrosis Conference.

Is RPMI 2650 a Suitable In Vitro Nasal Model for Drug Transport Studies?

The evaluation of new intranasal medications requires the development of in vitro cell model suitable for high-throughput screening studies. The aim of a pharmacological model is to closely mimic the barrier properties of human nasal mucosa that will influence drug pharmacokinetics. In this context, the human nasal cell line RPMI 2650 has been investigated over these last years. Although the initial studies tended to demonstrate strong physiological correlations between RPMI 2650 cells and nasal mucosa, the variability of experimental designs does not allow a clear comparison of actual data. Thereby, the standardization of cell culture parameters is crucial to obtain a stronger reproducibility and increase the relevance of data. Indeed, RPMI 2650 barrier properties are heavily dependent of cell culture conditions, especially of the physiological air-liquid interface that strengthen the expression of both tight junction proteins and drug transporters. Conversely, cell culture medium and insert composition showed a minor impact on the four key parameters of a nasal barrier. Despite the recent advances in the physiological characterization of RPMI 2650 model, only limited pharmacological data are available concerning the involvement of drug transporters in drug bioavailability. The deployment of standardized bi-directional permeability studies using reference compounds is required to determine the relevance of RPMI 2650 model in the field of drug transport studies.

Improved Transnasal Transport and Brain Uptake of Tizanidine HCl-Loaded Thiolated Chitosan Nanoparticles for Alleviation of Pain

The aim of this study was to prepare and characterize thiolated chitosan (TC) nanoparticles (NPs) of tizanidine HCl (TZ) and to evaluate its transport across monolayer of RPMI 2650 cells (Human nasal septum carcinoma cell line) followed by assessment of their pharmacokinetic and pharmacodynamic attributes, after intranasal (i.n.) administration. Chitosan was thiolated by carbodiimide method and thiolation was confirmed qualitatively and quantitatively. NPs were prepared using ionotropic gelation and evaluated for mucoadhesion using sheep nasal mucosa for drug permeation and cytotoxicity using monolayer of RPMI 2650 cells. Drug biodistribution was evaluated after technetium-99m labeling, visualized using gamma camera, and evaluated pharmacodynamically by measuring antinociceptive activity in mice. High mucoadhesion and permeation of drug were observed for TC NPs with least toxicity to nasal epithelial cells. Brain uptake and antinociceptive effect of the drug were significantly enhanced after thiolation of chitosan. This concludes that TC NPs, after i.n. administration, show significant increase in the mucoadhesion, reduction in cytotoxicity, enhanced permeation across cells monolayer, higher TZ brain uptake, and considerable increase in antinociceptive activity of TZ in mice. These features make TC an interesting polymer for demonstrating appreciable improvement of transnasal permeation of hydrophilic drugs, such as TZ, known to have limited permeation across blood-brain barrier.

Preparation and evaluation of spray-dried hyaluronic acid microspheres for intranasal delivery of fexofenadine hydrochloride

Hyaluronic acid (HA)-based microspheres containing PEG 6000 and/or sodium taurocholate (NaTC) were prepared by the spray-drying method for nasal delivery of fexofenadine hydrochloride (HCl). Their physicochemical properties including particle size and drug contents were determined, while their morphology examined by a scanning electron microscope (SEM). The effects of the solubilizer (PEG 6000) and the permeation enhancer (NaTC) on the in vitro release characteristics of fexofenadine ·HCl were observed. Moreover, the in vitro permeation of fexofenadine ·HCl was determined using the human nasal cell (HNE) monolayers cultured on Transwell ® inserts. After nasal administration, fexofenadine ·HCl in rabbit plasma was determined by the LC–MS/MS system. Fexofenadine ·HCl-loaded microspheres were of spherical shape with 20–30 μm mean diameter. The loading efficiency was about 95%. In vitro release of fexofenadine ·HCl from the microspheres was significantly increased with the addition of PEG 6000. Although NaTC did not alter the in vitro release of drug from the microspheres, its addition further increased the in vitro permeation of fexofenadine ·HCl across the HNE cell monolayers. Moreover, the bioavailability of fexofenadine ·HCl after nasal administration of the microsphere formulation to rabbits was increased up to about 48% while that of the control solution was only about 3%. These results indicated that the HA microsphere formulation could further be developed into a clinically useful nasal delivery system of fexofenadine ·HCl.

An improved primary human nasal cell culture for the simultaneous determination of transepithelial transport and ciliary beat frequency

Abstract Objectives The aim was to establish a preclinical in-vitro system of the nasal mucosa for the simultaneous evaluation of nasal absorption and effects on ciliary activity. Methods Human nasal epithelial cells were grown in collagen-coated transport inserts with transparent polyethylene terephthalate membranes (3 μm). Transepithelial transport and ciliary beat frequency values were measured every 15 min for 1 h. Key findings The apparent permeability coefficients (Papp) for atenolol (mainly paracellular transport) and propranolol (transcellular transport) amounted to 0.1 ± 0.1 and 23.7 ± 0.6 × 10−6 cm/s, respectively, illustrating that the system can be used to discriminate between high permeability and low permeability compounds. Transport of talinolol (substrate for the P-glycoprotein efflux carrier) did not reveal polarity (0.3 ± 0.2 and 0.2 ± 0.1 × 10−6 cm/s for absorptive and secretory transport, respectively) and was not affected by verapamil (10 μm), suggesting the absence of P-glycoprotein in the nasal cell culture. No significant effects of atenolol, propranolol and talinolol on ciliary beat frequency were observed (98 ± 20% of the control condition after 60 min). Chlorocresol significantly decreased the ciliary activity but this decrease was not accompanied by effects on the transepithelial transport of atenolol, propranolol and talinolol. Conclusions A new system was developed which offers possibilities as a fast screening tool for studying the potential of compounds for nasal drug administration, since permeability and a possible cilio-toxic effect can be assessed simultaneously.

Rescue of Functional F508del Cystic Fibrosis Transmembrane Conductance Regulator by Vasoactive Intestinal Peptide in the Human Nasal Epithelial Cell Line JME/CF15

F508del is the most common cystic fibrosis-causing mutation that induces early degradation and poor trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels to the apical membrane of epithelial cells. Our previous work in bronchial serous cells showed that vasoactive intestinal peptide (VIP) stimulation of the VPAC(1) receptor enhances CFTR-dependent chloride secretion by increasing its membrane insertion by a protein kinase C (PKC)-dependent pathway. In the present study, we investigated the effect of VIP on F508del-CFTR activity and membrane insertion in the human nasal epithelial cell line JME/CF15, which also expresses the VPAC(1) receptor. At reduced temperature (27 degrees C), which rescues F508del-CFTR trafficking, acute stimulation by VIP of rescued F508del-CFTR channels was protein kinase A (PKA)- and PKC-dependent. One hour of treatment with VIP strongly increased F508del-CFTR activity, with iodide efflux peaks three times higher than with untreated cells. At 37 degrees C, VIP-treated cells, but not untreated controls, showed significant iodide efflux peaks that were sensitive to the CFTR inhibitor 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (CFTR(inh)-172). Immunostaining, biotinylation assays, and Western blots confirmed a VIP-induced maturation and membrane insertion of F508del-CFTR at 37 degrees C. The corrector effect of VIP was abolished by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamidedihydrochloride (H89), whereas Galpha(s) stimulation by cholera toxin significantly increased F508del-CFTR trafficking. On the other hand, membrane localization, but not maturation, of F508del-CFTR was significantly reduced by the PKC inhibitor bisindolylmaleimide X and the G(i/o) protein inhibitor pertussis toxin. VIP treatment had no effect on intracellular calcium or proteasome activity. These results indicate that, in human nasal cells, VIP rescues trafficking and membrane insertion of functional F508del-CFTR channels at physiological temperature by stimulating both PKA- and PKC-dependent pathways.

An improved primary human nasal cell culture for the simultaneous determination of transepithelial transport and ciliary beat frequency

The aim was to establish a preclinical in-vitro system of the nasal mucosa for the simultaneous evaluation of nasal absorption and effects on ciliary activity. Human nasal epithelial cells were grown in collagen-coated transport inserts with transparent polyethylene terephthalate membranes (3 mum). Transepithelial transport and ciliary beat frequency values were measured every 15 min for 1 h. The apparent permeability coefficients (P(app)) for atenolol (mainly paracellular transport) and propranolol (transcellular transport) amounted to 0.1 +/- 0.1 and 23.7 +/- 0.6 x 10(-6) cm/s, respectively, illustrating that the system can be used to discriminate between high permeability and low permeability compounds. Transport of talinolol (substrate for the P-glycoprotein efflux carrier) did not reveal polarity (0.3 +/- 0.2 and 0.2 +/- 0.1 x 10(-6) cm/s for absorptive and secretory transport, respectively) and was not affected by verapamil (10 muM), suggesting the absence of P-glycoprotein in the nasal cell culture. No significant effects of atenolol, propranolol and talinolol on ciliary beat frequency were observed (98 +/- 20% of the control condition after 60 min). Chlorocresol significantly decreased the ciliary activity but this decrease was not accompanied by effects on the transepithelial transport of atenolol, propranolol and talinolol. A new system was developed which offers possibilities as a fast screening tool for studying the potential of compounds for nasal drug administration, since permeability and a possible cilio-toxic effect can be assessed simultaneously.