Sperm of both mammals and invertebrates move toward specific sites in the female reproductive tract. However, molecular mechanisms for sperm to follow directional cues are unknown. Here, we report genetic analysis of Drosophila Pkd2 at 33E3 (Pkd2, CG6504), which encodes a Ca(2+)-activated, nonselective cation channel homologous to the human Pkd2 autosomal dominant polycystic kidney disease (ADPKD) gene. The PKD2 family of genes has been implicated in sensory responses through protein localization on primary cilia of epithelia and neurons. In renal tubules, cilium-associated PKD2 appears to mediate Ca(2+) influx in response to fluid flow, and the loss of fluid sensation probably contributes to cyst growth and ADPKD. Sperm tails or flagella are specialized cilia essential for movement. Drosophila Pkd2 is abundantly associated with the tail and the acrosome-containing head region of mature sperm. Targeted disruption of Pkd2 results in male sterility without affecting spermatogenesis. The mutant sperm are motile but fail to swim into the storage organs in the female. Rare mutant sperm that reach the storage organs are able to fertilize the egg and produce viable progeny. Our data demonstrate that the Drosophila PKD2 cation channel operates in sperm for directional movement inside the female reproductive tract.

Among the multiple organ disorders caused by the severe acute respiratory syndrome coronavirus (SARS-CoV), acute lung failure following atypical pneumonia is the most serious and often fatal event. We hypothesized that two of the hydrophilic structural coronoviral proteins (S and E) would regulate alveolar fluid clearance by decreasing the cell surface expression and activity of amiloride-sensitive epithelial sodium (Na(+)) channels (ENaC), the rate-limiting protein in transepithelial Na(+) vectorial transport across distal lung epithelial cells. Coexpression of either S or E protein with human alpha-, beta-, and gamma-ENaC in Xenopus oocytes led to significant decreases of both amiloride-sensitive Na(+) currents and gamma-ENaC protein levels at their plasma membranes. S and E proteins decreased the rate of ENaC exocytosis and either had no effect (S) or decreased (E) rates of endocytosis. No direct interactions among SARS-CoV E protein with either alpha- or gamma-ENaC were indentified. Instead, the downregulation of ENaC activity by SARS proteins was partially or completely restored by administration of inhibitors of PKCalpha/beta1 and PKCzeta. Consistent with the whole cell data, expression of S and E proteins decreased ENaC single-channel activity in oocytes, and these effects were partially abrogated by PKCalpha/beta1 inhibitors. Finally, transfection of human airway epithelial (H441) cells with SARS E protein decreased whole cell amiloride-sensitive currents. These findings indicate that lung edema in SARS infection may be due at least in part to activation of PKC by SARS proteins, leading to decreasing levels and activity of ENaC at the apical surfaces of lung epithelial cells.

Davis IC, Xu A, Gao Z, Hickman-Davis JM, Factor P, Sullender WM, Matalon S. Respiratory syncytial virus induces insensitivity to ␤-adrenergic agonists in mouse lung epithelium in vivo. Am J Physiol Lung Cell Mol Physiol 293: L281-L289, 2007. First published April 13, 2007; doi:10.1152/ajplung.00458.2006 is the most common cause of bronchiolitis in infants and children worldwide. We wished to determine whether intratracheal administration of ␤-agonists improved alveolar fluid clearance (AFC) across the distal respiratory epithelium of RSV-infected mice. Following intranasal infection with RSV strain A2, AFC was measured in anesthetized, ventilated BALB/c mice by instillation of 5% BSA into the dependent lung. We found that direct activation of protein kinase A by forskolin or 8-bromo-cAMP increased AFC at day 2 after infection with RSV. In contrast, short-and long-acting ␤-agonists had no effect at either day 2 or day 4. Insensitivity to ␤-agonists was not a result of elevated plasma catecholamines or lung epithelial cell ␤-adrenergic receptor degradation. Instead, RSV-infected mice had significantly higher levels of phosphorylated PKC in the membrane fractions of their lung epithelial cells. In addition, insensitivity to ␤-agonists was mediated in a paracrine fashion by KC (the murine homolog of CXCL8) and reversed by inhibition of either PKC or G protein-coupled receptor kinase 2 (GRK2). These results indicate that insufficient response to ␤-agonists in RSV may be caused, at least in part, by impaired ␤-adrenergic receptor signaling, as a consequence of GRK2-mediated uncoupling of ␤-adrenergic receptors from adenylyl cyclase. paramyxovirus; protein kinase C; G protein-coupled receptor kinase 2; CXCL8 RESPIRATORY SYNCYTIAL VIRUS (RSV) is the most common cause of lower respiratory tract disease in infants and children worldwide (44), is a frequent initiator of acute asthma exacerbations in young children, and has a disease impact comparable to that of nonpandemic influenza A in the elderly (8). Approximately 2-3% of all cases of RSV bronchiolitis result in severe hypoxia or a need for parenteral fluid supplementation that necessitates hospitalization (44). ␤-agonists are frequently used to treat RSV bronchiolitis, primarily because of their perceived ability to relax airway smooth muscle and cause bronchodilation, with the ultimate aim of alleviating hypoxemia. However, it is not clear that these drugs are clinically effective: meta-analyses have shown little or no overall benefit, regardless of viral bronchiolitis severity (15,20). Their lack of efficacy has not been explained, although it has often been ascribed to difficulties associated with drug delivery to the small airways of young infants, particularly in the presence of bronchoconstriction and inflammatory exudates or airway obstruction (28). ␤-agonists increase total body oxygen consumption, thereby increasing oxygen demands in infants hospitalized for respiratory compromise, and can potentially exacerbate ventilation-perfusion mismatch by inducing vaso...

Surfactant protein D (SP-D) is an important effector of innate immunity. We have previously shown that SP-D accumulates at sites of acute bacterial infection and neutrophil infiltration, a setting associated with the release of reactive species such as peroxynitrite. Incubation of native SP-D or trimeric SP-D lectin domains (NCRDs) with peroxynitrite resulted in nitration and nondisulfide cross-linking. Modifications were blocked by peroxynitrite scavengers or pH inactivation of peroxynitrite, and mass spectroscopy confirmed nitration of conserved tyrosine residues within the C-terminal neck and lectin domains. Mutant NCRDs lacking one or more of the tyrosines allowed us to demonstrate preferential nitration of Tyr314 and the formation of Tyr228-dependent cross-links. Although there was no effect of peroxynitrite or tyrosine mutations on lectin activity, incubation of SP-D dodecamers or murine lavage with peroxynitrite decreased the SP-D-dependent aggregation of lipopolysaccharide-coated beads, supporting our hypothesis that defective aggregation results from abnormal cross-linking. We also observed nitration, cross-linking of SP-D, and a significant decrease in SP-D-dependent aggregating activity in the lavage of mice acutely exposed to nitrogen dioxide. Thus, modification of SP-D by reactive oxygen-nitrogen species could contribute to alterations in the structure and function of SP-D at sites of inflammation in vivo.

IL-1R-associated kinase (IRAK)-1 is a critical mediator of TLR/IL-1R-induced activation of the transcription factor NF-κB. We previously described that a commonly occurring IRAK-1 variant haplotype, containing amino acid changes from serine to phenylalanine at position 196 and from leucine to serine at position 532, is associated with increased activation of NF-κB in LPS-stimulated neutrophils from patients with sepsis-induced acute lung injury and also higher mortality and more severe clinical outcomes in such patients. To investigate the underlying molecular mechanisms, we examined the ability of wild-type and variant IRAK-1 to modulate NF-κB activation. We found increased NF-κB transcriptional activity and expression of NF-κB-dependent proinflammatory cytokines in IL-1β-stimulated IRAK-1-deficient cells transfected with variant IRAK-1 as compared with IRAK-1 wild type. IκB-α degradation was faster and p65 phosphorylation more prolonged after IL-1β stimulation in cells expressing the IRAK-1 variant. However, IL-1-induced activation of MAPKs and nuclear translocation of NF-κB are comparable in both IRAK-1 variant- and IRAK-1 wild-type-expressing cells. Autophosphorylation of the IRAK-1 variant is greater than that found with wild-type IRAK-1. Additionally, variant IRAK-1 has greater interaction with TNFR-associated factor 6 than does wild-type IRAK-1. The enhanced activity of variant IRAK-1 appeared to be due to the alteration at aa 532, with only minimal effects being associated with change at aa 196. These results demonstrate that variant IRAK-1 is associated with alterations in multiple intracellular events that are likely to contribute to increased NF-κB activation and inflammatory responses in individuals with this IRAK-1 haplotype.