Soluble adenylyl cyclase mediates hydrogen peroxide-induced changes in epithelial barrier function

BACKGROUND: Elevated H(2)O(2) levels are associated with inflammatory diseases and H(2)O(2) exposure is known to disrupt epithelial barrier function, leading to increased permeability and decreased electrical resistance. In normal human bronchial epithelial (NHBE) cells, fully differentiated at the air liquid interface (ALI), H(2)O(2) activates an autocrine prostaglandin pathway that stimulates transmembrane adenylyl cyclase (tmAC) as well as soluble adenylyl cyclase (sAC), but the role of this autocrine pathway in H(2)O(2)-mediated barrier disruption is not entirely clear. METHODS: To further characterize the mechanism of H(2)O(2)-induced barrier disruption, NHBE cultures were treated with H(2)O(2) and evaluated for changes in transepithelial resistance and mannitol permeability using agonist and inhibitors to dissect the pathway. RESULTS: A short (<10 min) H(2)O(2) treatment was sufficient to induce resistance and permeability changes that occurred 40 min to 1 h later and the changes were partially sensitive to EP1 but not EP4 receptor antagonists. EP1 receptors were localized to the apical compartment of NHBE. Resistance and permeability changes were sensitive to inhibition of sAC but not tmAC and were partially blocked by PKA inhibition. Pretreatment with a PLC inhibitor or an IP3 receptor antagonist reduced changes in resistance and permeability suggesting activation of sAC occurred through increased intracellular calcium. CONCLUSION: The data support an important role for prostaglandin activation of sAC and PKA in H(2)O(2)-induced barrier disruption. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12931-016-0329-4) contains supplementary material, which is available to authorized users

Calcium Influx Mediates the Voltage-Dependence of Sperm Entry into Sea Urchin Eggs

Sperm entry was monitored in voltage-clamped sea urchin eggs following insemination in a variety of artificial seawaters. In regular seawater, maintaining the membrane potential at increasingly negative values progressively inhibits sperm entry. Reducing [Ca2+]o relieves the inhibition, shifting the sperm entry vs voltage relationship toward more negative potentials. Raising [Ca2+]o shifts the relationship in the other direction. Large changes in [Na+]o or [Mg2+]o do not affect sperm entry although changing [Na+]o dramatically changes the currents following sperm attachment. Applying one of seven different calcium channel blockers or replacing Ca2+ with Ba2+ or Sr2+ or microinjecting calcium chelators into the cytoplasm relieves the block to sperm entry at negative potentials. We conclude that the block to sperm entry at negative potentials is mediated by calcium which crosses the membrane and acts at an intracellular site

H 2

Cystic fibrosis transmembrane conductance regulator (CFTR) activity is essential for the maintenance of airway surface liquid depth, and therefore mucociliary clearance. Reactive oxygen species, increased during inflammatory airway diseases, alter CFTR activity. Here, H(2)O(2) levels in the surface liquid of normal human bronchial epithelial cultures differentiated at the air–liquid interface were estimated, and H(2)O(2)-mediated changes in CFTR activity were examined. In Ussing chambers, H(2)O(2)-induced anion currents were sensitive to the CFTR inhibitors CFTR(inh)172 and GlyH-101. These currents were absent in cells from patients with cystic fibrosis. Responses to greater than 500 μM H(2)O(2) were transient. Cyclooxygenase inhibitors blocked the H(2)O(2) response, as did EP1 and EP4 receptor antagonists. A multidrug-resistant protein (MRP) inhibitor and short hairpin RNA directed against MRP4 blocked H(2)O(2) responses. EP1 and EP4 agonists mimicked H(2)O(2) in both control and MRP4 knockdown cells. Thus, H(2)O(2) activates the synthesis, export, and binding of prostanoids via EP4 and, interestingly, EP1 receptors in normal, differentiated human airway epithelial cells to activate cyclic adenosine monophosphate pathways that in turn activate CFTR channels in the apical membrane

Additional file 1: Figure S1. of Soluble adenylyl cyclase mediates hydrogen peroxide-induced changes in epithelial barrier function

H2O2 inhibition of forskolin-stimulated IS C. NHBE cultures were mounted in Ussing chambers and treated with either 10 μM forskolin or 1mM H2O2 followed by 10 μM forskolin. Panel a, representative traces of forskolin-induced CFTR activity with (red trace) and without (black trace) H2O2 pre-treatment. Panel b, ISC values with (treated) and without (control) H2O2 pre-treatment (mean ± s.e.m., n = 9 lung donors). H2O2 treatment reduced the forskolin-stimulated ISC by approximately 50 %. Figure S2. Recovery of resistance and forskolin-stimulated ISC after H2O2 treatment. NHBE cultures were exposed to apical 1 mM H2O2 for 1h. After removal of H2O2 the cultures were returned to the CO2 incubator for 23h and then placed in Ussing chambers for measurement of both resistance and forskolin-stimulated IS C (time point 24h). Panel a, ISC before and 23h after treatment are shown (mean ± s.e.m., n = 3 lung donors). Forskolin-stimulated ISC currents returned to normal within 23h after treatment. Panel b, Epithelial resistance before and 23h after treatment are shown (mean ± s.e.m., n = 3 lung donors). Resistance returns to normal within 23h after treatment. (DOCX 633 kb

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

Hemitiroidectomía mediante videoendoscopia, experiencia en el Hospital "Hermanos Ameijeiras": an experience from "Hermanos Ameijeiras" Clinical Surgical Hospital Hemithyroidectomy by videoendoscopy

INTRODUCCIÓN: se presentan las 4 primeras pacientes operadas de hemitiroidectomía videoendoscópica en el Hospital "Hermanos Ameijeiras", para introducir una técnica que elimine la cicatriz en el cuello de los(as) pacientes que necesitan un tratamiento quirúrgico por una enfermedad tiroidea. MÉTODOS: se escogieron 4 pacientes con nódulos benignos del tiroides menores de 3 cm, remitidas para tratamiento quirúrgico por el servicio de endocrinología de nuestro centro, de enero a junio de 2009. Se les explicó en detalles el proceder y firmaron un consentimiento informado para participar en el estudio. RESULTADOS: en las 4 pacientes se pudo realizar la operación planificada, el promedio de tiempo quirúrgico fue de 2,9 h. El efecto cosmético fue mayor en las pacientes 2, 3 y 4 al no quedar ninguna cicatriz en el cuello. La laringoscopia posoperatoria demostró movilidad normal de las cuerdas vocales en las 4 pacientes. CONCLUSIONES: la cirugía videoendoscópica del tiroides es una realidad que no podemos obviar, y realmente tiene un único impacto demostrado hasta el momento actual, que es la posibilidad de eliminar la cicatriz residual en el cuello que tanto preocupa a los(as) pacientes jóvenes.INTRODUCTION: these are the cases of the first patients operated on of Hemithyroidectomy by videoendoscopy in "Hermanos Ameijeiras" Clinical Surgical Hospital to introduce a technique eliminating the neck scar of patients needing surgical treatment by thyroid disease. METHODS: four patients presenting with benign thyroid nodules referred to surgical treatment by Endocrinology service of our institution from January to June, 2009. Procedure was explained in details and they signed the informed consent to participate in study. RESULTS: in four patients it was possible to perform the planned operation; the surgical time average was of 2, 9 hr. Cosmetic effect was greater in patients 2, 3 and 4 without neck scar. Postoperative laryngoscopy showed a normal motility of vocal cords in the four patients. CONCLUSIONS: thyroid videoendoscopy surgery is a reality that cannot be obviated and with a unique impact proved until present time that is the possibility to eliminate the neck residual scar so worrying for young patients

Transforming Growth Factor-β1 and Cigarette Smoke Inhibit the Ability of β 2

Chronic bronchitis, caused by cigarette smoke exposure, is characterized by mucus hypersecretion and reduced mucociliary clearance (MCC). Effective MCC depends, in part, on adequate airway surface liquid. Cystic fibrosis transmembrane conductance regulator (CFTR) provides the necessary osmotic gradient for serosal to mucosal fluid transport through its ability to both secrete Cl(−) and regulate paracellular permeability, but CFTR activity is attenuated in chronic bronchitis and in smokers. β(2)-adrenergic receptor (β(2)-AR) agonists are widely used for managing chronic obstructive pulmonary disease, and can activate CFTR, stimulate ciliary beat frequency, and increase epithelial permeability, thereby stimulating MCC. Patients with chronic airway diseases and cigarette smokers demonstrate increased transforming growth factor (TGF)-β1 signaling, which suppresses β(2)-agonist–mediated CFTR activation and epithelial permeability increases. Restoring CFTR function in these diseases can restore the ability of β(2)-agonists to enhance epithelial permeability. Human bronchial epithelial cells, fully redifferentiated at the air–liquid interface, were used for (14)C mannitol flux measurements, Ussing chamber experiments, and quantitative RT-PCR. β(2)-agonists enhance epithelial permeability by activating CFTR via the β(2)-AR/adenylyl cyclase/cAMP/protein kinase A pathway. TGF-β1 inhibits β(2)-agonist–mediated CFTR activation and epithelial permeability enhancement. Although TGF-β1 down-regulates both β(2)-AR and CFTR mRNA, functionally it only decreases CFTR activity. Cigarette smoke exposure inhibits β(2)-agonist–mediated epithelial permeability increases, an effect reversed by blocking TGF-β signaling. β(2)-agonists enhance epithelial permeability via CFTR activation. TGF-β1 signaling inhibits β(2)-agonist–mediated CFTR activation and subsequent increased epithelial permeability, potentially limiting the ability of β(2)-agonists to facilitate paracellular transport in disease states unless TGF-β1 signaling is inhibited

IFN-γ-mediated reduction of large-conductance, Ca 2+

Effective mucociliary clearance (MCC) depends in part on adequate airway surface liquid (ASL) volume to maintain an appropriate periciliary fluid height that allows normal ciliary activity. Apically expressed large-conductance, Ca(2+)-activated, and voltage-dependent K(+) (BK) channels provide an electrochemical gradient for Cl(−) secretion and thus play an important role for adequate airway hydration. Here we show that IFN-γ decreases ATP-mediated apical BK activation in normal human airway epithelial cells cultured at the air-liquid interface. IFN-γ decreased mRNA levels of KCNMA1 but did not affect total protein levels. Because IFN-γ upregulates dual oxidase (DUOX)2 and therefore H(2)O(2) production, we hypothesized that BK inactivation could be mediated by BK oxidation. However, DUOX2 knockdown did not affect the IFN-γ effect on BK activity. IFN-γ changed mRNA levels of the BK β-modulatory proteins KCNMB2 (increased) and KCNMB4 (decreased) as well as leucine-rich repeat-containing protein (LRRC)26 (decreased). Mallotoxin, a BK opener only in the absence of LRRC26, showed that BK channels lost their association with LRRC26 after IFN-γ treatment. Finally, IFN-γ caused a decrease in ciliary beating frequency that was immediately rescued by apical fluid addition, suggesting that it was due to ASL volume depletion. These data were confirmed with direct ASL measurements using meniscus scanning. Overexpression of KCNMA1, the pore-forming subunit of BK, overcame the reduction of ASL volume induced by IFN-γ. Key experiments were repeated in cystic fibrosis cells and showed the same results. Therefore, IFN-γ induces mucociliary dysfunction through BK inactivation