Functional and molecular characterization of an anion exchanger in airway serous epithelial cells

Serous cells secreteCl and HCO₃ and play an importantrole in airway function. Recent studies suggest that aCl/HCO₃ anion exchanger (AE) maycontribute to Cl secretion by airway epithelial cells.However, the molecular identity, the cellular location, and thecontribution of AEs to Cl secretion in serous epithelialcells in tracheal submucosal glands are unknown. The goal of thepresent study was to determine the molecular identity, the cellularlocation, and the role of AEs in the function of serous epithelialcells. To this end, Calu-3 cells, a human airway cell line with aserous-cell phenotype, were studied by RT-PCR, immunoblot, andelectrophysiological analysis to examine the role of AEs inCl secretion. In addition, the subcellular location of AEproteins was examined by immunofluorescence microscopy. Calu-3 cellsexpressed mRNA and protein for AE2 as determined by RT-PCR and Westernblot analysis, respectively. Immunofluorescence microscopy identified AE2 in the basolateral membrane of Calu-3 cells in culture and rattracheal serous cells in situ. InCl/HCO₃/Na⁺-containingmedia, the 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate(CPT-cAMP)-stimulated short-circuit anion current (I_sc) was reduced by basolateral but not byapical application of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid(50 µM) and 4,4'-dinitrostilbene-2,2'-disulfonic acid [DNDS (500 µM)], inhibitors of AEs. In the absence of Na⁺ in thebath solutions, to eliminate the contributions of the Na⁺/HCO₃ andNa⁺/K⁺/2Cl cotransporters toI_sc, CPT-cAMP stimulated a small DNDS-sensitive I_sc. Taken together with previous studies, theseobservations suggest that a small component of cAMP-stimulatedI_sc across serous cells may be referable toCl secretion and that uptake of Cl acrossthe basolateral membrane may be mediated by AE2.

     

Peptide toxin blockers of voltage-sensitive K+ channels: inotropic effects on diaphragm.

Agents that block many types of K+ channels (e.g., the aminopyridines) have substantial inotropic effects in skeletal muscle. Specific blockers of ATP-sensitive and Ca2+-activated K+ channels, on the other hand, do not, or minimally, alter the force of nonfatigued muscle, consistent with a predominant role for voltage-gated K+ channels in regulating muscle force. To test this more directly, we examined the effects of peptide toxins, which in other tissues specifically block voltage-gated K+ channels, on rat diaphragm in vitro. Twitch force was increased in response to alpha-, beta-, and gamma-dendrotoxin and tityustoxin Kalpha (17 +/- 6, 22 +/- 5, 42 +/- 14, and 13 +/- 5%; P < 0.05, < 0.01, < 0.05, < 0.05, respectively) but not in response to delta-dendrotoxin or BSA (in which toxins were dissolved). Force during 20-Hz stimulation was also increased significantly by alpha-, beta-, and gamma-dendrotoxin and tityustoxin Kalpha. Among agents, increases in twitch force correlated with the degree to which contraction time was prolonged (r = 0.88, P < 0.02). To determine whether inotropic effects could be maintained during repeated contractions, muscle strips underwent intermittent 20-Hz train stimulation for a duration of 2 min in presence or absence of gamma-dendrotoxin. Force was significantly greater with than without gamma-dendrotoxin during repetitive stimulation for the first 60 s of repetitive contractions. Despite the approximately 55% higher value for initial force in the presence vs. absence of gamma-dendrotoxin, the rate at which fatigue occurred was not accelerated by the toxin, as assessed by the amount of time over which force declined by 25 and 50%. These data suggest that blocking voltage-activated K+ channels may be a useful therapeutic strategy for augmenting diaphragm force, provided less toxic blockers of these channels can be found.     

Roles of vaccinia virus genes E3L and K3L and host genes PKR and RNase L during intratracheal infection of C57BL/6 mice

The importance of the 2'-5' oligoadenylate synthetase (OAS)/RNase L and double-stranded RNA (dsRNA)-dependent protein kinase (PKR) pathways in host interferon induction resulting from virus infection in response to dsRNA has been well documented. In poxvirus infections, the interactions between the vaccinia virus (VV) genes E3L and K3L, which target RNase L and PKR, respectively, serve to prevent the induction of the dsRNA-dependent induced interferon response in cell culture. To determine the importance of these host genes in controlling VV infections, mouse single-gene knockouts of RNase L and PKR and double-knockout mice were studied following intratracheal infection with VV, VVΔK3L, or VVΔE3L. VV caused lethal disease in all mouse strains. The single-knockout animals were more susceptible than wild-type animals, while the RNase L(-/-) PKR(-/-) mice were the most susceptible. VVΔE3L infections of wild-type mice were asymptomatic, demonstrating that E3L plays a critical role in controlling the host immune response. RNase L(-/-) mice showed no disease, whereas 20% of the PKR(-/-) mice succumbed at a dose of 10(8) PFU. Lethal disease was routinely observed in RNase L(-/-) PKR(-/-) mice inoculated with 10(8) PFU of VVΔE3L, with a distinct pathology. VVΔK3L infections exhibited no differences in virulence among any of the mouse constructs, suggesting that PKR is not the exclusive target of K3L. Surprisingly, VVΔK3L did not disseminate to other tissues from the lung. Hence, the cause of death in this model is respiratory disease. These results also suggest that an unanticipated role of the K3L gene is to facilitate virus dissemination.     

Phosphorylation of Vesicular Stomatitis Virus In Vivo and In Vitro

The structural protein, NS, of purified vesicular stomatitis virus (VSV) is a phosphoprotein. In infected cells phosphorylated NS is found both free in the cytoplasm and as part of the viral ribonucleoprotein (RNP) complex containing both the 42S RNA and the structural proteins L, N, and NS, indicating that phosphorylation occurs as an early event in viral maturation. VSV contains an endogenous protein kinase activity, probably of host region, which catalyzes the in vitro phosphorylation of the viral proteins NS, M, and L, but not of N or G. The phosphorylated sites on NS appear to be different in the in vivo and in vitro reactions, and are differentially sensitive to alkaline phosphatase. After removal of the membrane components of purified VSV with a dextran-polyethylene glycol two-phase separation, the kinase activity remains tightly associated with the viral RNP. However, viral RNP isolated from infected cells shows only a small amount of kinase activity. The protein kinase enzyme appears to be a cellular contaminant of purified VSV because an activity from the uninfected cell extract can phosphorylate in vitro the dissociated viral proteins NS and M. The virion-associated activity may be derived either from the cytoplasm or the plasma membrane of the host cell since both of these cellular components contain protein kinase activity similar to that found in purified VSV.     

A survey of intracellular Na+ and K+ of various normal,transformed, and tumor cells

Intracellular concentrations of Na⁺ and K⁺ of various normal, transformed, and tumor cell cultures were analyzed by atomic absorption spectrophotometry. In all of the cultures analyzed there were markedly different concentrations in the transformed and tumor cells when compared to their normal counterparts. Although increased Na⁺ was often observed, there were no definitive correlations between absolute ion concentrations, or Na⁺:K⁺ ratios, and cell transformation.     

Bathymetric distribution and feeding habits of two sympatric cheilodactylid fishes at Miyake-jima,Japan

Habitats including depth, diurnal activity patterns, and diets of the two cheilodactylid fishesCheilodactylus zonatus andC. zebra were studied at Igaya Bay, Miyake-jima, Japan. Both species occurred at nearly equal densities in identical habitats and depths. They preyed on benthonic organisms from early morning to shortly before sunset, when feeding activities decreased remarkably and intraspecific social behavior greatly increased. Gut analyses showed a high degree of dietary overlap, but proportions of food items in their diets differed slightly.C. zonatus was more of a food generalist thanC. zebra, showing a higher niche breadth value.C. zebra tends to feed on epifauna, especially gammaridean amphipods and decapods, whileC. zonatus takes both epifauna and infauna including polychaetes.     

Comparison of Clark's presence-absence test and the membrane filter method for coliform detection in potable water samples

A total of 2,601 water samples from six different water systems were tested for coliform bacteria by Clark's presence-absence (P-A) test and by the membrane filter (MF) method. There was no significant difference in the fraction of samples positive for coliform bacteria for any of the systems tested. It was concluded that the two tests are equivalent for monitoring purposes. However, 152 samples were positive for coliform bacteria by the MF method but negative by the P-A test, and 132 samples were positive by the P-A test but negative by the MF method. Many of these differences for individual samples can be explained by random dispersion of bacteria in subsamples when the coliform density is low. However, 15 samples had MF counts greater than 3 and gave negative P-A results. The only apparent explanation for most of these results is that coliform bacteria were present in the P-A test bottles but did not produce acid and gas. Two other studies have reported more samples positive by Clark's P-A test than by the MF method.