Zoltan Sutto

Soluble adenylyl cyclase is localized to cilia and contributes to ciliary beat frequency regulation via production of cAMP

Ciliated airway epithelial cells are subject to sustained changes in intracellular CO2/HCO3 − during exacerbations of airway diseases, but the role of CO2/HCO3 −-sensitive soluble adenylyl cyclase (sAC) in ciliary beat regulation is unknown. We now show not only sAC expression in human airway epithelia (by RT-PCR, Western blotting, and immunofluorescence) but also its specific localization to the axoneme (Western blotting and immunofluorescence). Real time estimations of [cAMP] changes in ciliated cells, using FRET between fluorescently tagged PKA subunits (expressed under the foxj1 promoter solely in ciliated cells), revealed CO2/HCO3 −-mediated cAMP production. This cAMP production was specifically blocked by sAC inhibitors but not by transmembrane adenylyl cyclase (tmAC) inhibitors. In addition, this cAMP production stimulated ciliary beat frequency (CBF) independently of intracellular pH because PKA and sAC inhibitors were uniquely able to block CO2/HCO3 −-mediated changes in CBF (while tmAC inhibitors had no effect). Thus, sAC is localized to motile airway cilia and it contributes to the regulation of human airway CBF. In addition, CO2/HCO3 − increases indeed reversibly stimulate intracellular cAMP production by sAC in intact cells.

Regulation of human airway ciliary beat frequency by intracellular pH.

pHi affects a number of cellular functions, but the influence of pHi on mammalian ciliary beat frequency (CBF) is not known. CBF and pHi of single human tracheobronchial epithelial cells in submerged culture were measured simultaneously using video microscopy (for CBF) and epifluorescence microscopy with the pH‐sensitive dye BCECF. Baseline CBF and pHi values in bicarbonate‐free medium were 7.2 ± 0.2 Hz and 7.49 ± 0.02, respectively (n= 63). Alkalization by ammonium pre‐pulse to pHi 7.78 ± 0.02 resulted in a 2.2 ± 0.1 Hz CBF increase (P < 0.05). Following removal of NH4Cl, pHi decreased to 7.24 ± 0.02 and CBF to 5.8 ± 0.1 Hz (P < 0.05). Removal of extracellular CO2 to change pHi resulted in similar CBF changes. Pre‐activation of cAMP‐dependent protein kinase (10 μm forskolin), broad inhibition of protein kinases (100 μm H‐7), inhibition of PKA (10 μm H‐89), nor inhibition of phosphatases (10 μm cyclosporin + 1.5 μm okadaic acid) changed pHi‐mediated changes in CBF, nor were they due to [Ca2+]i changes. CBF of basolaterally permeabilized human tracheobronchial cells, re‐differentiated at the air–liquid interface, was 3.9 ± 0.3, 5.7 ± 0.4, 7.0 ± 0.3 and 7.3 ± 0.3 Hz at basolateral i.e., intracellular pH of 6.8, 7.2, 7.6 and 8.0, respectively (n= 18). Thus, intracellular alkalization stimulates, while intracellular acidification attenuates human airway CBF. Since phosphorylation and [Ca2+]i changes did not seem to mediate pHi‐induced CBF changes, pHi may directly act on the ciliary motile machinery.

Decreased Soluble Adenylyl Cyclase Activity in Cystic Fibrosis Is Related to Defective Apical Bicarbonate Exchange and Affects Ciliary Beat Frequency Regulation

Human airway cilia contain soluble adenylyl cyclase (sAC) that produces cAMP upon HCO3−/CO2 stimulation to increase ciliary beat frequency (CBF). Because apical HCO3− exchange depends on cystic fibrosis transmembrane conductance regulator (CFTR), malfunctioning CFTR might impair sAC-mediated CBF regulation in cells from patients with cystic fibrosis (CF). By Western blot, sAC isoforms are equally expressed in normal and CF airway epithelial cells, but CBF decreased more in CF than normal cells upon increased apical HCO3−/CO2 exposure in part because of greater intracellular acidification from unbalanced CO2 influx (estimated by 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence). Importantly, ciliated cell-specific cAMP production (estimated by FRET fluorescence ratio changes of tagged cAMP-dependent protein kinase (PKA) subunits expressed under a ciliated cell-specific promoter) in response to increased apical HCO3−/CO2 perfusion was higher in normal compared with CF cells. Inhibition of bicarbonate influx via CFTR (CFTRinh172) and inhibition of sAC (KH7) and PKA activation (H89) led to larger CBF declines in normal cells, now comparable with changes seen in CF cells. These inhibitors also reduced FRET changes in normal cells to the level of CF cells with the expected exception of H89, which does not prevent dissociation of the fluorescently tagged PKA subunits. Basolateral permeabilization and subsequent perfusion with HCO3−/CO2 rescued CBF and FRET changes in CF cells to the level of normal cells. These results suggest that CBF regulation by sAC-produced cAMP could be impaired in CF, thereby possibly contributing to mucociliary dysfunction in this disease, at least during disease exacerbations when airway acidification is common.

Calcium‐mediated, purinergic stimulation and polarized localization of calcium‐sensitive adenylyl cyclase isoforms in human airway epithelia

Purinergic stimulation of human airway epithelia results in a prolonged increase in ciliary beat frequency that depends on calcium‐mediated cAMP production [Lieb, T., Wijkstrom Frei, C., Frohock, J.I., Bookman, R.J. and Salathe, M. (2002) Prolonged increase in ciliary beat frequency after short‐term purinergic stimulation in human airway epithelial cells. J. Physiol. (Lond.) 538, 633–646]. Here, fully differentiated human airway epithelial cells in culture are shown to express calcium‐stimulated transmembrane adenylyl cyclase (tmAC) isoforms (types 1, 3, and 8) by reverse transcription polymerase chain reaction. Immunohistochemistry of tracheal sections and fully differentiated airway epithelial cell cultures revealed polarized expression of these tmACs, with types 1 and 8 localized to the apical membrane and thus at the position required for ciliary regulation. Real‐time, ciliated‐cell specific cAMP production by tmACs upon apical, purinergic stimulation with UTP was confirmed using fluorescent energy resonance transfer between fluorescently tagged PKA subunits.

Spontaneous Pneumomediastinum After Kidney Transplantation: Case Report

Spontaneous pneumomediastinum is a rare condition with nonspecific signs and symptoms. A 39-year-old underwent cadaver kidney transplantation. After an uncomplicated operation, progressive dyspnea of unknown origin developed. Findings at chest radiography suggested pneumomediastinum, which was confirmed at computed tomography. Esophageal or tracheal injury was ruled out. The rapidly developing atelectasis of the left lung necessitated urgent bronchoscopy, which revealed occlusion of the left main bronchus. After removal of the occluding mucus plug, the clinical symptoms immediately improved, and the spontaneous pneumomediastinum resolved within 3 days. Asymptomatic increase in airway secretions in patients receiving peritoneal dialysis may result in mucus plug formation during general anesthesia, which can cause spontaneous pneumomediastinum.

Seasonal changes of lower respiratory tract infections in lung transplant recipients during the first post‐transplant year: The Hungarian experience

After lung transplantation (LuTX) a high level of immunosuppression is needed to prevent rejection of the graft. Together with earlier colonization by pathogens, immunosuppression makes recipients more susceptible to infections, especially during the first postoperative year. As seasonality of lower respiratory tract infections (LRTI) is well‐known in chronic lung diseases, we assessed seasonal changes of pathogen spectrum and number of infections in the first postoperative year in LuTX recipients.

Severe mixed sleep apnea after bilateral lung transplantation in a cystic fibrosis patient: a case report.

BACKGROUND Lung transplantation is the only treatment for end-stage lung disease in selected patients. After lung transplantation, patient recovery is often slow owing to severe underlying diseases in the patient producing hypoxemia before, during, and after surgery, as well as infections and rejection episodes. Postoperative breathing and ventillatory disorders are also associated with diaphragmatic dysfunction and/or phrenic nerve damage. METHODS Herein we have reported a case of a 35-year-old man undergoing bilateral lung transplantation owing to worsening of chronic respiratory failure from cystic fibrosis. After uncomplicated surgery, weaning was delayed due to nighttime dyspnea and hypoxemia attributed to diaphragm dysfunction. After improvement of diaphragm function, the symptoms persisted, requiring noninvasive nocturnal ventilatory support. Polysomnography confirmed severe mixed sleep apnea. RESULTS Effective treatment with noninvasive bi-level positive airway pressure spontaneous/timed mode (BiPAP S/T) ventilation during the nights rendered the patient symptom free. Polysomnography confirmed successful treatment. CONCLUSION Disordered breathing while sleeping is common after solid organ transplantation. BiPAP S/T ventilator therapy was effective to the treat dominantly central sleep apnea in our patient.