The mechanism of the elevation of intracellular free Ca2+ ([Ca2+]i) induced by a single sperm in eggs of the sea urchin Lytechinus variegatus was investigated. Simultaneous measurements of [Ca2+]i, and of the activation current, were carried out on eggs microinjected with Ca Green-1 or Ca Green dextran, and voltage clamped at -20 mV. The microinjection of 0.5 to 1.0 mg/ml heparin (MW 6000) or pentosan polysulfate (MW 3000), final intracellular concentration, causes a concentration-dependent inhibition in all parameters of the sperm-induced elevation of [Ca2+]i and the phase 2 calcium-activated cation current (Ip). For each: (1) the onset is delayed; (2) the rate of change is slowed; and (3) the peak amplitude attained is diminished. In some experiments at the higher concentrations, the microinjected polysulfates cause the complete suppression of the sperm-induced elevation of [Ca2+]i and Ip. The entry of multiple sperm overcomes the inhibitory effects of the polysulfates. Our data suggest that inositol 1,4,5-trisphosphate is the primary mechanism responsible for the sperm-induced release of Ca2+ from intracellular stores.

No abstract

To examine cholinergic signal transduction pathways that modulate ciliary beat frequency (CBF), cultured ovine tracheal epithelial cells were imaged using a combination of phase-contrast (CBF) and fluorescence (Ca2+) microscopy techniques. In single cells, acetylcholine (ACh) transiently increased CBF and intracellular Ca2+ concentration ([Ca2+]i), mainly by Ca2+ release from internal stores, with a small delayed contribution from Ca2+ influx. Nicotinic agonists did not alter CBF or [Ca2+]i, whereas atropine blocked the ACh-stimulated transients, consistent with the involvement of muscarinic receptors. 4-Diphenylacetoxy-N-methylpiperidine methiodide was approximately 100 times more potent than pirenzepine in inhibiting the ACh-induced [Ca2+]i peaks, suggesting that the receptor is a pharmacologically defined (M3) subtype. Interestingly, after depletion of intracellular Ca2+ stores by thapsigargin, ACh caused a rapid transient decrease in both CBF and [Ca2+]i, again with an antagonist profile of M3 receptors. We conclude that activation of M3 muscarinic receptors initiates specific signaling pathways that act simultaneously to increase and decrease [Ca2+]i and CBF.

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 2 and regulate paracellular permeability, but CFTR activity is attenuated in chronic bronchitis and in smokers. b 2 -adrenergic receptor (b 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)-b1 signaling, which suppresses b 2 -agonist-mediated CFTR activation and epithelial permeability increases. Restoring CFTR function in these diseases can restore the ability of b 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. b 2 -agonists enhance epithelial permeability by activating CFTR via the b 2 -AR/adenylyl cyclase/cAMP/ protein kinase A pathway. TGF-b1 inhibits b 2 -agonist-mediated CFTR activation and epithelial permeability enhancement. Although TGF-b1 down-regulates both b 2 -AR and CFTR mRNA, functionally it only decreases CFTR activity. Cigarette smoke exposure inhibits b 2 -agonist-mediated epithelial permeability increases, an effect reversed by blocking TGF-b signaling. b 2 -agonists enhance epithelial permeability via CFTR activation. TGF-b1 signaling inhibits b 2 -agonist-mediated CFTR activation and subsequent increased epithelial permeability, potentially limiting the ability of b 2 -agonists to facilitate paracellular transport in disease states unless TGF-b1 signaling is inhibited.

Adenylyl cyclases are generally thought to be transmembrane-, G-protein-, and forskolin-responsive proteins, but a nontransmembrane, soluble adenylyl cyclase (sAC) 2 has been identified (1). Mammalian sAC is not activated by G-proteins or forskolin (1) but by HCO 3 Ϫ /CO 2 in a pH-independent manner (2, 3) and by Ca 2ϩ , which synergizes with HCO 3 Ϫ (4). sAC expression has been described in many human tissues (5). We have shown that it is expressed in the airway epithelium, where it represents the only known adenylyl cyclase localized to cilia (6). sAC is important for flagellar beating in sperm (7-9), and we have shown its importance for regulating ciliary beating in human airway epithelia via cAMP production upon stimulation with HCO 3 Ϫ /CO 2 (6). There are different sources of luminal bicarbonate in the airways: it can be secreted from submucosal glands and ciliated cells (10) and can also be produced de novo from CO 2 and H 2 O by locally secreted carbonic anhydrase (11). Two proteins responsible for transporting HCO 3 Ϫ into cells, Slc26a9 (12, 13) and CFTR (14), have been described in the apical membrane of airway epithelial cells. Slc26a9 is mainly a chloride channel with very low bicarbonate permeability (12); thus, the major apical HCO 3 Ϫ exchange in human airways occurs likely through CFTR (14) or is dependent on it.In 1989, the CFTR gene was linked to cystic fibrosis (15). Initial attempts to localize the protein in the airways were based on mRNA in situ hybridization and pinpointed expression mainly to submucosal gland acini (16). Significant expression of CFTR was more recently also shown in the apical membrane of ciliated cells from healthy human beings, whereas CFTR was absent in the apical membrane of cells from CF patients homozygous for the ⌬F508 mutation (17). Expression of human CFTR (driven by the ciliated cell-specific promoter foxj1) in the trachea of a CF mouse model confirmed targeting of CFTR to the apical membrane of ciliated cells while restoring forskolinstimulated chloride secretion (18). Electrolyte conductance through CFTR is usually associated with chloride transport, but bidirectional conductance of bicarbonate through CFTR has also been demonstrated and is thought to be important in the airway and in pancreatic ducts (14, 19 -21). Regulation of HCO 3 Ϫ transport through CFTR has been related to sAC in Calu3 cells, an airway epithelial cell line (22), and in corneal endothelium (23), but there are no reports of the influence of diminished bicarbonate transport through CFTR on the activity of sAC in primary airway epithelial cells, especially as it * This work was supported, in whole or in part, by National Institutes of Health

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 H2O2 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.

BACKGROUND AND PURPOSEH2O2 is widely understood to regulate intracellular signalling. In airway epithelia, H2O2 stimulates anion secretion primarily by activating an autocrine PGE2 signalling pathway via EP4 and EP1 receptors to initiate cytic fibrosis transmembrane regulator (CFTR)-mediated Cl − secretion. This study investigated signalling downstream of the receptors activated by H2O2. EXPERIMENTAL APPROACHAnion secretion by differentiated bronchial epithelial cells was measured in Ussing chambers during stimulation with H2O2, an EP4 receptor agonist or β2-adrenoceptor agonist in the presence and absence of inhibitors of ACs and downstream effectors. Intracellular calcium ([Ca 2+ ]I) changes were followed by microscopy using fura-2-loaded cells and PKA activation followed by FRET microscopy. KEY RESULTSTransmembrane adenylyl cyclase (tmAC) and soluble AC (sAC) were both necessary for H2O2 and EP4 receptor-mediated CFTR activation in bronchial epithelia. H2O2 and EP4 receptor agonist stimulated tmAC to increase exchange protein activated by cAMP (Epac) activity that drives PLC activation to raise [Ca 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. Effective airway clearance relies on the regulation of ciliary beat frequency (CBF), adequate airway surface liquid (ASL) volume, and correct mucus properties. Mucus hydration and thus the viscosity of mucus depend, in part, on the movement of water through the airway epithelium to the mucosal surface via the coordinated activities of ion channels. An important player in this regulation is the cystic fibrosis transmembrane conductance regulator (CFTR) that passes chloride (and other anions) while regulating the epithelial Na 1 channel to balance osmolarity (1). CFTR and CBF are both regulated through increases in cyclic adenosine monophosphate (cAMP) and protein kinase A activity (2-4). Thus, mediators that stimulate adenylyl cyclase are expected to result in improved airway clearance by increasing chloride secretion and CBF.Airway epithelia actively synthesize H 2 O 2 via the nicotinamide adenine dinucleotide phosphate-reduced (NADPH) dual oxidases Duox1 and Duox2, as reviewed by Fischer (5), presumably for the use of the lactoperoxidase host defense against infection (6-9). Duox1 and Duox2 are regulated by cytokines (10-12) and bacterial products (10,13,14) to increase H 2 O 2 synthesis to high levels (10). Stimulation with interferon-g increased H 2 O 2 synthesis levels 30-fold when measured in air-liquid interface (ALI) cultures of normal human airway epithelial cells (10), leading to 10 mM H 2 O 2 in washes used for assays. The experiments presented here address ASL H 2 O 2 and show that it is present at high concentrations.In addition to its role in stimulating lactoperoxidase host defense, H 2 O 2 may also regulate the innate defense related to mucociliary clearance. In the bronchial submucosal adenocarcinoma cell line Calu-3, CFTR activity is increased by exposure of the airway epithelium to exogenous H 2 O 2 (15, 16). In cell lines, the effect of H 2 O 2 on CFTR is reported to occur through increases in transmembrane adenylyl cyclase activity and through the activation of two K 1 channels (15), most likely KCNQ1 and KCNN4, which are located in the basolateral membrane. These channels are believed to provide a driving force for apical Cl 2 release (17, 18). Yet the mechanisms by which H 2 O 2 leads to the activation of the cAMP pathway are not understood. Although both Calu-3 cells (19) and bovine tracheal epithelia (20) have been shown to respond to isop...