Differentiation of ciliated cells in the terminal bronchioles of neonatal calves

The bronchiolar ciliated cells are exquisitely sensitive to injury caused by infection or irritation of the airways. The mechanism by which bronchiolar ciliated cells are renewed following injury or during the normal course of differentiation is still debated. The present study aimed at recognizing the progenitor cell population for bronchiolar ciliated cells during early neonatal life of calves and to demonstrate the course of events occurs during its differentiation into ciliated cells. Scanning electron microscopy of the terminal bronchiolar epithelium revealed two distinct cell types namely ciliated and non-ciliated cells. Transmission electron microscopy revealed ciliated, non-ciliated (Clara), intermediate and basal cells. At least two categories of intermediate cells could be distinguished: intermediate cells with abundant glycogen and variable numbers of organelles; intermediate cells with little glycogen, large numbers of polyribosomes, and variable numbers of basal bodies. We conclude that: (1) both bronchiolar non-ciliated and basal cells serve as progenitors for the bronchiolar ciliated cells; (2) differentiation of ciliated cell from the non-ciliated one involves a transitional cell in which glycogen is lost, polyribosomes are synthesized before the synthesis of basal bodies and cilia.     

Hypoxic enlarged mitochondria protect cancer cells from apoptotic stimuli

It is well established that cells exposed to the limiting oxygen microenvironment (hypoxia) of tumors acquire resistance to chemotherapy, through mechanisms not fully understood. We noted that a large number of cell lines showed protection from apoptotic stimuli, staurosporine, or etoposide, when exposed to long‐term hypoxia (72 h). In addition, these cells had unusual enlarged mitochondria that were induced in a HIF‐1‐dependent manner. Enlarged mitochondria were functional as they conserved their transmembrane potential and ATP production. Here we reveal that mitochondria of hypoxia‐induced chemotherapy‐resistant cells undergo a HIF‐1‐dependent and mitofusin‐1‐mediated change in morphology from a tubular network to an enlarged phenotype. An imbalance in mitochondrial fusion/fission occurs since silencing of not only the mitochondrial fusion protein mitofusin 1 but also BNIP3 and BNIP3L, two mitochondrial HIF‐targeted genes, reestablished a tubular morphology. Hypoxic cells were insensitive to staurosporine‐ and etoposide‐induced cell death, but the silencing of mitofusin, BNIP3, and BNIP3L restored sensitivity. Our results demonstrate that some cancer cells have developed yet another way to evade apoptosis in hypoxia, by inducing mitochondrial fusion and targeting BNIP3 and BNIP3L to mitochondrial membranes, thereby giving these cells a selective growth advantage. J. Cell. Physiol. 222: 648–657, 2010. © 2009 Wiley‐Liss, Inc.     

Nitric oxide production and absorption in trachea, bronchi, bronchioles, and respiratory bronchioles of humans

Different volumesof dead-space gas were collected and analyzed for nitric oxide (NO)content, either immediately after inspiration or after a period ofbreath holding on clean air or NO mixtures. This allowed calculation ofNO equilibrium, NO production, and NO absorption. In seven young,healthy, adult nonsmokers, the mean NO equilibrium values in parts perbillion (ppb) were 56 ± 11 (SE) in the trachea, 37 ± 6 in thebronchi, 21 ± 3 in the bronchioles, and 16 ± 2 in therespiratory bronchioles. At any given NO concentration, the NOabsorption rate (in nl/min) equaled the NO concentration (in ppb) timesA (the absorption coefficient inl/min). A values (in l/min) were 0.11 ± 0.01 in the trachea, 0.17 ± 0.04 in the bronchi, 0.66 ± 0.09 in the bronchioles, and 1.35 ± 0.32 in the respiratorybronchioles. NO equilibrium concentrations and production rates in one74-yr-old subject were three to five times as high as those found inthe young subjects. Mouth equilibrium NO concentrations were 3 and 6 parts per million in two subjects who had oral production rates of 6 and 23 nl/min, respectively. In conclusion, production and absorptionof NO occur throughout the first 450 ml of the airways.

     

Functionally distinct pools of actin in secretory cells

Acid secretion by the gastric parietal cell is controlledthrough movement of vesicles containing the proton pump, theH⁺-K⁺-ATPase (HK). We have usedlatrunculin B (Lat B), which binds to monomeric actin, to investigateactin turnover in the stimulated parietal cell. In isolated gastricglands, relatively high concentrations of Lat B were required toinhibit acid accumulation (ED₅₀ ~70 µM). Culturedparietal cells stimulated in the presence of low Lat B (0.1-1µM) have reduced lamellipodia formation and some aberrant punctatephalloidin-stained structures, but translocation of HK and vacuolarswelling appeared unaffected. High Lat B (10-50 µM) resulted ingross changes in actin organization (punctate phalloidin-stainedstructures throughout the cell and nucleus) and reduced translocationof HK and vacuolar swelling. Resting parietal cells treated with highLat B showed minor effects on morphology and F-actin staining. Ifresting cells treated with high Lat B were washed immediately beforestimulation, they exhibited a normal stimulated morphology. These datasuggest distinct pools of parietal cell actin: a pool highlysusceptible to Lat B primarily involved in motile function of culturedcells; and a Lat B-resistant pool, most likely microvillar filaments,that is essential for secretion. Furthermore, the stimulation processappears to accentuate the effects of Lat B, most likely through Lat Bbinding to monomer actin liberated by the turnover of the motile actinfilament pool.

     

Separation and characterization of basal and secretory cells from the rat trachea by flow cytometry

Basal and secretory cells have been separated as highly enriched viable populations from single-cell suspensions of rat tracheal epithelial cells. Isolation of the populations was achieved by preparation of a cell suspension and separation by flow cytometry using contour maps generated from 2 degrees and 90 degrees light scatter signals. Flow cytometric analysis of cells showed 10% of the whole preparation were cells in SG2M phase of the cell cycle. The secretory cells accounted for 86% of these cycling cells; the remainder were accounted for by the basal cells. Culture of sorted populations of basal and secretory cells in serum free defined medium showed that basal cells had a lower (0.6%) colony-forming efficiency than secretory cells (3.4%). Significant differences in blue auto-fluorescence, Hoechst 33342 uptake, and lectin staining were apparent between basal and secretory cells. These results suggest that the secretory cell rather than the basal cell is primarily the cell type involved in maintenance of the normal tracheal epithelium. Secretory cells are greater in number, have a higher proliferative potential, and greater metabolic capability. Because of these traits they may be a critical cell at risk from damage by environmental agents.     

The distribution of cationized ferritin receptors on ciliated epithelial cells of rat trachea

The interaction of tracheal cilia with the biphasic mucus layer covering the surface of the mammalian respiratory tract may be influenced by many cell surface coat components including those having an overall negative charge. In order to assess the distribution of ciliary anionic sites, cationized ferritin (CF) was used to label the surface of rat tracheal epithelium. If pieces of trachea were fixed with 3% glutaraldehyde and treated with CF at low (L) (0.08 mg/ml), medium (M) (0.32 mg/ml PBS), or high (H) (0.64 mg/ml PBS) concentrations, the label was distributed evenly over the entire external surface of the ciliary membrane at all concentrations. Unfixed tracheal tissue was also treated with L, M, and H CF for 1 or 5 min at 4 degrees C in order to minimize lateral redistribution of CF receptors. To ensure accessibility of the cell surface to CF the samples were agitated thoroughly during exposure. Exposure for 1 min to L, M, and H CF resulted in a light binding of ferritin particles on all portions of the ciliary membrane with occasional areas of multilayered binding distributed randomly on the ciliary shaft. When unfixed trachea was treated with CF for 5 min at 4 degrees C, CF binding was similar except heavier and more uniform. In no instance was there any preferential binding of CF to the ciliary tips at any of the concentrations used. Moreover, as indicated by the CF binding pattern at L concentrations, high density negative charges are present over almost the entire surface of the cilium. These results suggest that, unlike the ciliary membrane of other organs such as oviduct, negatively charged cell surface coat molecules are present on all areas of the ciliary membrane of rat tracheal epithelia.     

Dynamic image analysis applied to the study of ciliary beat on cultured ciliated epithelial cells from rabbit trachea

We have developed an automated image analysis method to study the ciliary beat frequency of ciliated cells of the primary culture from rabbit trachea. The ciliated outgrowth image is digitized and the variation in optical density is automatically calculated for each selected area of interest. 32 measurements of ciliary beat frequency are, in this way, calculated simultaneously in 6 min. With this reliable device, some studies on baseline frequency of control culture have been carried out. There was no variation in the mean frequencies of ciliated cells of the primary culture of different tracheas in our culture conditions. Moreover, the values of ciliary beat frequency at the starting point of the outgrowth were similar to those at the periphery of the outgrowth. There is nevertheless a slow decrease in frequencies versus the duration of culture. We have also established that the frequency of ciliary beat of some cells fluctuates in a periodic pattern whereas the majority of the ciliated population beat in a stable way. The image analysis process allows us to perform a cartography of frequencies on the video display. It also allows us to have access to the frequency of one cilium. Our method therefore seems to be reliable and furthermore simple in the evaluation of the potential effect of inhaled toxic compounds on ciliated cells of mammalian respiratory tract.     

CCSP regulates cross talk between secretory cells and both ciliated cells and macrophages of the conducting airway

Pulmonary host defense employs a combination of biochemical and biophysical activities to recognize, inactivate, and mediate clearance of environmental agents as well as modulate the overall response to such challenge. Dysregulation of the inflammatory arm of this response is associated with chronic lung diseases (CLD) including cystic fibrosis and chronic obstructive lung disease. Although mechanisms mediating immunoregulation are incompletely characterized, decrements in levels of the nonciliated secretory cell product Clara cell secretory protein (CCSP) in numerous CLD and identification of proinflammatory state in mice homozygous for a null allele of the CCSP gene (CCSP-/-) suggest a central role for the nonciliated secretory cell in this process. In an effort to determine the molecular basis for immunoregulatory defects associated with CCSP deficiency, we utilized difference gel electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight to compare the proteomes of wild-type and CCSP-/- mice. We demonstrate a shift in the isoelectric point of the immunomodulatory protein annexin A1 (ANXA1) to more acidic isoforms in CCSP-/- mice. Similar ANXA1 mRNA and protein abundance in wild-type and CCSP-/- tissue and identical localization of ANXA1 protein to alveolar macrophages and the ciliary bed of ciliated cells demonstrated that CCSP deficiency was associated exclusively with altered posttranslational modification of ANXA1. These results suggest that both long- and short-range paracrine signaling between nonciliated secretory cells and cells of the immune system and epithelium impact modification of cell type-specific proteins and implicate nonciliated secretory cells in a regulatory axis that might integrate critical aspects of host defense.     

An ultrastructural study of primary cilia,abnormal cilia and ciliary knobs from the ciliated cells of the guinea-pig trachea

Summary Single primary cilia are found in developing as well as mature ciliated cells of guinea-pig tracheal epithelium. A few biciliated cells were observed, and in a rare case one cell had developed four such processes. Primary cilia are characterized by a 9 + 0 microtubular arrangement near the base, while a transition to an 8 + 1 pattern occurs at a slightly more distal position. Spokes are lacking, and dynein arms are absent or incompletely developed. The function, if any, of primary cilia remains unknown.In the population of the motile 9 + 2 cilia atypical forms are very rare, i.e. <0.1%. Of the various abnormalities cilia with supernumary microtubules are most common. Only one atypical basal body was observed. Although some of the aberrant forms undoubtedly are non-motile, their very low number suggests that they have no practical influence on the muco-ciliary clearance.Local extrusions of the ciliary membrane, here named ciliary knobs, are believed to be fixation artefacts. It is suggested that they represent circumscribed regions of the ciliary membrane which are sensitive to changes in the environmental osmotic pressure.     

Sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary

The distribution of three proteins discharged by regulated exocytosis--growth hormone (GH), prolactin (PRL), and secretogranin II (SgII)--was investigated by double immunolabeling of ultrathin frozen sections in the acidophilic cells of the bovine pituitary. In mammotrophs, heavy PRL labeling was observed over secretory granule matrices (including the immature matrices at the trans Golgi surface) and also over Golgi cisternae. In contrast, in somatotrophs heavy GH labeling was restricted to the granule matrices; vesicles and tubules at the trans Golgi region showed some and the Golgi cisternae only sparse labeling. All somatotrophs and mammotrophs were heavily positive for GH and PRL, respectively, and were found to contain small amounts of the other hormone as well, which, however, was almost completely absent from granules, and was more concentrated in the Golgi complex, admixed with the predominant hormone. Mixed somatomammotrophs (approximately 26% of the acidophilic cells) were heavily positive for both GH and PRL. Although admixed within Golgi cisternae, the two hormones were stored separately within distinct granule types. A third type of granule was found to contain SgII. Spillage of small amounts of each of the three secretory proteins into granules containing predominantly another protein was common, but true intermixing (i.e., coexistence within single granules of comparable amounts of two proteins) was very rare. It is concluded that in the regulated pathway of acidophilic pituitary, cell mechanisms exist that cause sorting of the three secretory proteins investigated. Such mechanisms operate beyond the Golgi cisternae, possibly at the sites where condensation of secretion products into granule matrices takes place.     

Purification of mitochondria and secretory granules isolated from pancreatic beta cells using Percoll and Sephacryl S-1000 superfine

Mitochondria and secretory granules were isolated from beta-cell-rich pancreatic islets of ob/ob mice. Crude fractions obtained by differential centrifugation were subjected to centrifugation on isotonic Percoll. The gradient medium was removed from the resulting fractions by gel filtration on Sephacryl S-1000 Superfine. When compared to the crude fractions, the purified mitochondrial fraction exhibited a 4.5-fold increase in citrate synthase activity, a 70% reduction of lysosomal arylsulfatase, and a 40% decrease of contamination with granular insulin. In the purified secretory granule fraction, the insulin content was as high as 60% of the total protein (albumin standard) with arylsulfatase unchanged and no detectable citrate synthase activity.     

Identification of a voltage-gated potassium channel in gerbil hippocampal mitochondria

Transient cerebral ischemia is known to induce endogenous mechanisms that can prevent or delay neuronal injury, such as the activation of mitochondrial potassium channels. However, the molecular mechanism of this effect remains unclear. In this study, the single-channel activity was measured using the patch-clamp technique of the mitoplasts isolated from gerbil hippocampus. In 70% of all patches, a potassium-selective current with the properties of a voltage-gated Kv-type potassium channel was recorded with mean conductance 109 ± 6 pS in a symmetrical solution. The channel was blocked at negative voltages and irreversibly by margatoxin, a specific Kv1.3 channel inhibitor. The ATP/Mg²⁺ complex and Ca²⁺ ions had no effect on channel activity. Additionally, agitoxin-2, a potent inhibitor of voltage-gated potassium channels, had no effect on mitochondrial channel activity. This observation suggests that in contrast to surface membrane channels, the mitochondrial voltage-gated potassium channel could have a different molecular structure with no affinity to agitoxin-2. Western blots of gerbil hippocampal mitochondria and immunohistochemistry on gerbil brain sections confirmed the expression of the Kv1.3 protein in mitochondria. Our findings indicate that gerbil brain mitochondria contain a voltage-gated potassium channel that can influence the function of mitochondria in physiological and pathological conditions and that has properties similar to the surface membrane Kv1.3 channel.     

Transdifferentiation of outgrowth cells and cultured epithelial cells from swine trachea

Summary The morphologic and functional properties of explant out-growth cells and epithelial cells isolated from swine trachea epithelium by proteolysis were examined. A mixed population of ciliated, serous, and basal cells, obtained from out-growths, from proteolysis of trachea epithelium, and from unattached explants in organ culture, all yielded cell cultures that were composed almost entirely of mucus-secreting cells. When the cells were grown in primary or secondary culture on a modified collagen matrix in supplemented HAM:DMEM (1:1) medium they expressed a mucus-secreting phenotype with numerous mucus granules at various stages of maturation and incorporated [³H]GlcN and³⁵SO₄ into secreted mucin glycoproteins. Results obtained in these studies suggest that extensive transdifferentiation of ciliated and serous cells to mucus-secreting cells occurs after the release and during subsequent attachment and culture. Ciliated cells containing mucus granules were seen in various stages of cilia resorption. Basal cells containing mucus granules were also frequently observed. The number of mucus-secreting cells and the synthesis of mucin glycoproteins increased dramatically with time of attachment and culture, whereas cell proliferation, population doubling time of 72 h, and incorporation of [³H]thymidine into DNA increased much more slowly. The number of mucus-secreting cells correlated closely with the level of secretion of mucin glycoproteins. Taken collectively, these studies help to elucidate the transdifferentiation process, which dramatically increases the number of mucus-secreting cells after disruption and release of epithelial cells from swine tracheobronchial epithelium. A similar mechanism involving disruption of the extracellular matrix may be involved in the stimulation of hypersecretion of mucus and mucin glycoproteins by chemical and infections irritants.     

Expression of ASIC2 in ciliated cells and stereociliated cells

Acid-sensing ion channel 2 (ASIC2) plays a role as a mechanorecptor and acid receptor in the peripheral and central nervous systems. However, several recent studies have suggested that ASIC2 is expressed in several organs, in addition to the nervous system. We have examined the expression and distribution of ASIC2 in rat ciliated cells (trachea and oviduct) and stereociliated cells (epididymis, Corti organ, and ampullary crest) by immunohistochemistry and transmission electron microscopy (TEM). Immunohistochemistry revealed that ASIC2 was expressed in both ciliated cells and stereociliated cells, but the localization differed between these cell types. In ciliated cells, ASIC2 was coexpressed with a cilial marker (acetylated tubulin). In stereociliated cells stained with a stereocilial marker (phalloidin), ASIC2 was observed in the cell body. Observation by TEM suggested that ASIC2 expression was present at the apical side of the cilial membrane in ciliated cells and at the apical side of the cell body in stereociliated cells. This study thus indicates that the proton receptor ASIC2 is expressed in both ciliated and stereociliated cells.