However, the effects of the PDE4 inhibitor roflumilast on smoke-induced mucociliary dysfunction have not been fully explored. Methods Primary normal human bronchial epithelial cells (NHBE) from non-smokers, cultured at the air-liquid interface (ALI) were used for most experiments. beat frequency (CBF) employing infrared differential interference contrast microscopy. Chloride conductance was measured in Ussing chambers and CFTR expression was quantified with qPCR. Results While treatment with 100 nM roflumilast experienced little effect alone, it increased intracellular cAMP upon activation with forskolin and albuterol in cultures exposed to cigarette smoke and in control conditions. cAMP baselines were lower in smoke-exposed cells. Roflumilast prolonged cAMP increases in smoke-exposed and control cultures. Smoke-induced reduction in functional, albuterol-mediated chloride conductance through CFTR was improved by roflumilast. ASL volumes also increased in smoke-exposed cultures in the presence of roflumilast while it did not in its absence. Cigarette smoke exposure decreased CBF, an effect rescued with roflumilast, particularly when used together with the long-acting ?-mimetic formoterol. Roflumilast also enhanced forskolin-induced CBF activation in ASL volume supplemented smoked and control cells, confirming the direct stimulatory effect of rising cAMP on ciliary function. In active smokers, CFTR mRNA expression was increased compared to non-smokers and ex-smokers. Roflumilast also increased CFTR mRNA levels in cigarette-smoke uncovered cell cultures. Conclusions Our results show that roflumilast can rescue smoke-induced mucociliary dysfunction by reversing decreased CFTR activity, augmenting ASL volume, and stimulating CBF, the latter particularly in combination with formoterol. As expected, CFTR mRNA expression was not indicative of apical CFTR function. promoter were utilized for FRET as explained previously [13]. Briefly, recombinant lentiviruses were constructed using the pRRLsinPPT.CMV.MCS.Wpre vector [16]. For the initial constructs, genes encoding the catalytic PKA subunit CAT and the regulatory PKA subunit RII, fused to the fluorescent proteins YFP and CFP, respectively [17], were cloned into the multiple cloning site downstream of the ciliated cell-specific promoter for single expression in ciliated cells [18]. Using calcium phosphate co-precipitation (Clontech Laboratories, Inc., Mountain View, CA, USA), lentiviruses were prepared by co-transfecting HEK 293?T cells with vector and packaging DNAs plasmids. Virus-containing medium was collected 48?h and 72?h later, concentrated by polyethylene glycol Rabbit Polyclonal to ELOVL4 (11?%) precipitation, and stored at ?80?C. An estimation of the computer virus titer was performed using the p24 HIV Antigen ELISA kit (PerkinElmer, Wellesley, MA, USA). Dedifferentiated cells were utilized for co-infection with both viral constructs. At the time of plating the cells on Transwells, computer virus was added at a ratio of 100?ng per 500,000 cells in bronchial epithelial growth medium (BEGM) containing polybrene (2?g/ml final concentration). The infection was carried out immediately, at 37?C in 5?% CO2. The following day, computer virus was removed, and BEGM was changed to ALI medium top and bottom until cells reached confluence, when an air flow liquid interface was created. Expression of the fluorescently tagged proteins was monitored using an inverted fluorescence microscope. Measurement of CBF and FRET in airway epithelial cells Fully differentiated NHBE cells cultured on 24?mm Transwell supports were placed in a customized, fully enclosed chamber, allowing independent perfusion of the apical and basolateral compartments. The chamber was mounted at room temperature around the stage of an upright Nikon E600fn microscope. Water was added on top of the closed chamber for use of a 63 water immersion objective with a numerical aperture of 1 1.0. FRET was measured as explained previously [13], with images acquired every 10s. CBF was recorded according to published methods [13, 19], using infrared differential interference contrast video microscopy. CBF and FRET were measured in real time and simultaneously in ciliated cells that expressed both fusion proteins. In addition, CBF was also RS 127445 recorded on an inverted Zeiss Axiovert without apical perfusion before and after apical DPBS supplementation. Ussing chamber experiments Snapwell filters made up of fully differentiated NHBE cells were rinsed with Krebs-Henseleit answer (KH), and then mounted in Ussing chambers (EasyMount Chambers; Physiologic Devices, San Diego, CA, USA) made up of KH in apical and basolateral chambers. Solutions were managed at 37?C by heated water overcoats, and were continuously bubbled with a 95?% room air flow / 5?% CO2 combination to maintain the pH at 7.4. To monitor short-circuit current (ISC), the transepithelial membrane potential was clamped at 0?mV with a six-channel voltage clamp (model VCC MC6; Physiologic Devices) using Ag/AgCl electrodes in agar bridges. Signals were digitized and recorded with DAQplot software (VVI Software, College Station, PA, USA) via a LabJack A/D converter (LabJack Corp., Lakewood, CO, USA). The input resistance of each filter was measured by the application of 1?mV bipolar pulses of 2-s duration. To eliminate any contribution to the Isc by epithelial sodium channels, 10?M amiloride was added to the apical. em p /em ? ?0.05 was accepted as significant. smoke and in control conditions. cAMP baselines were lower in smoke-exposed cells. Roflumilast prolonged cAMP increases in smoke-exposed and control cultures. Smoke-induced reduction in functional, albuterol-mediated chloride conductance through CFTR was improved by roflumilast. ASL volumes also increased in smoke-exposed cultures in the presence of roflumilast while it did not in its absence. Cigarette smoke exposure decreased CBF, an effect rescued with roflumilast, particularly when used together with the long-acting ?-mimetic formoterol. Roflumilast also enhanced forskolin-induced CBF activation in ASL volume supplemented smoked and control cells, confirming the direct stimulatory effect of rising cAMP on ciliary function. In active smokers, CFTR mRNA expression was increased compared to non-smokers and ex-smokers. Roflumilast also increased CFTR mRNA levels in cigarette-smoke uncovered cell cultures. Conclusions Our results show that roflumilast can rescue smoke-induced mucociliary dysfunction by reversing decreased CFTR activity, augmenting ASL volume, and stimulating CBF, the latter particularly in combination with formoterol. As expected, CFTR mRNA expression was not indicative of apical CFTR function. promoter were utilized for FRET as explained previously [13]. Briefly, recombinant lentiviruses were constructed using the pRRLsinPPT.CMV.MCS.Wpre vector [16]. For the initial constructs, genes encoding the catalytic PKA subunit CAT and the regulatory PKA subunit RII, fused to the fluorescent proteins YFP and CFP, respectively [17], were cloned into the multiple cloning site downstream of the ciliated cell-specific promoter for single expression in ciliated cells [18]. Using calcium phosphate co-precipitation (Clontech Laboratories, Inc., Mountain View, CA, USA), lentiviruses were prepared by co-transfecting HEK 293?T cells with vector and packaging DNAs plasmids. Virus-containing medium was collected 48?h and 72?h later, concentrated by polyethylene glycol (11?%) precipitation, and stored at ?80?C. An estimation of the computer virus titer was performed using the p24 HIV Antigen ELISA kit (PerkinElmer, Wellesley, MA, USA). Dedifferentiated cells were used for co-infection with both viral constructs. At the time of plating the cells on Transwells, virus was added at a ratio of 100?ng per 500,000 cells RS 127445 in bronchial epithelial growth medium (BEGM) containing polybrene (2?g/ml final concentration). The infection was done overnight, at 37?C in 5?% CO2. The following day, virus was removed, and BEGM was changed to ALI medium top and bottom until cells reached confluence, when an air liquid interface was created. Expression of the fluorescently tagged proteins was monitored using an inverted fluorescence microscope. Measurement of CBF and FRET in airway epithelial cells Fully differentiated NHBE cells cultured on 24?mm Transwell supports were placed in a customized, fully enclosed chamber, allowing independent perfusion of the apical and basolateral compartments. The chamber was mounted at room temperature on the stage of an upright Nikon E600fn microscope. Water was added on top of the closed chamber for use of a 63 water immersion objective with a numerical aperture of 1 1.0. FRET was measured as described previously [13], with images acquired every 10s. CBF was recorded according to published methods [13, 19], using infrared differential interference contrast video microscopy. CBF and FRET were measured in real time and simultaneously in ciliated cells that expressed both fusion proteins. In addition, CBF was also recorded on an inverted Zeiss Axiovert without apical perfusion before and after apical DPBS supplementation. Ussing chamber experiments Snapwell filters containing fully differentiated NHBE cells were rinsed with Krebs-Henseleit solution (KH), and then mounted in Ussing chambers (EasyMount Chambers; Physiologic Instruments, San Diego, CA, USA) containing KH in apical RS 127445 and basolateral chambers. Solutions were maintained at 37?C by heated water jackets, and were continuously bubbled with a 95?% room air / 5?% CO2 mixture to maintain RS 127445 the pH at 7.4. To monitor short-circuit current (ISC), the transepithelial membrane potential was clamped at 0?mV with a six-channel voltage clamp (model VCC MC6; Physiologic Instruments) using Ag/AgCl electrodes in agar bridges..
Prostanoid Receptors