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.

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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