Tracheal Membrane Research Articles

BRDU IMMUNOHISTOCHEMICAL OBSERVATION ON REGENERATION OF TRACHEAL EPITHELIA USING TISSUE CULTURE METHOD

The growth process of tracheal epithelia was observed by primary cell culture method using BrdU. Cells stained with BrdU, that showed mitotic activity, were nonciliary cells and BrdU was not found in ciliated (differentiated) cells. The dividing cells and the mature cells could be distinguished by BrdU. Thus, it was demonstrated that BrdU could be used as an index for investigating the growth process of basal cells. Tissue culture of the tracheal rings after mucosal injury was performed. On the third day post-injury, the cells stained with BrdU and the migrating epithelia were observed in the border region between the non-injury and injury site. On the seventh day post-injury, healing was completed. The tissue culture method using BrdU is useful for observing the healing process of the tracheal mucous membrane.

Submucosal tissues modulate the bioelectric properties of airway epithelium.

Exposure of isolated canine tracheal epithelium to acetylcholine (Ach) increased transepithelial conductance (GT) and short-circuit current (ISC). Qualitatively different responses were obtained when the epithelium was studied as a part of an intact posterior tracheal membrane that contained smooth muscle. Ach decreased GT, did not affect ISC, and induced a 2-fold increase in measured tension. In these tissues, baseline GT was enhanced compared with that of isolated epithelium. Indomethacin inhibited the Ach-induced responses of isolated epithelium but did not alter the responses of intact airway tissue. The epithelial origin of these electrophysiologic responses was confirmed in experiments that showed that denudation of intact tracheal tissue greatly increased base-line GT, eliminated ISC, abolished the electrophysiologic response to Ach, and enhanced Ach-induced smooth muscle constriction. The qualitatively different responses to Ach of intact tracheal membrane and epithelium alone suggest that the behavior of airway epithelia in the whole animal may be significantly different from that observed in conventional in vitro studies of isolated epithelia.

High-conductance Ca 2+-activated K + channels: Pharmacology and molecular characterization

High-conductance Ca2+-activated K+ channels (maxi-K channels) represent a family of proteins that display high selectivity and conductance for K+. With the discovery and purification of selective, high affinity, peptidyl probes for these channels, it has been possible to develop the molecular pharmacology of the maxi-K channel, and address the physiological role which these channels play in target tissues. Although molecular biology approaches have been successful in cloning other types of K+ channels, the molecular constituents of maxi-K channels are unknown. To gain insight into the structural composition of maxi-K channels, the ChTX receptor from bovine aortic and tracheal sarcolemmal membrane vesicles has been solubilized in functional form with digitonin, characterized, and purified to apparent homogeneity. Reconstitution experiments with these preparations demonstrate the presence of maxi-K channel activity with properties that are indistinguishable from those of the native channel. Studies are underway to determine sequence information for the identified ChTX receptor subunits in order to clone and express the maxi-K channel.

The Process of Recovery in a Case of Tracheal Injury Caused by Extubation

We have seen a case of tracheal injury caused by extubation. A 16-year-old boy who was sent to our hospital after a traffic accident was extubated by himself. Three day later, he coughed out a substance which was pathologically diagnosed as necrotic mucous membrane of the trachea. Tracheal fiberscopy revealed rubor, hemorrhage and discharge in the upper tracheal region. As the respiratory state and a chest X-ray revealed no pathological changes, a conservative therapy was chosen. We observed the recovery process with a fiberscope. Fourteen days later, the tracheal mucous membrane had healed up without complications.

N-alkyl-nor-tropine esters of 2-phenyl-cyclohexenic acids as new bronchodilator agents. Effect of structural and conformational modifications on affinity for muscarinic receptors

A series of N-alkyl-nor-tropine esters of 2-phenyl-2-cyclohexene-1-carboxylic, 2-phenyl-cyclohexanecarboxylic, and 2-phenyl-1-cyclohexene-1-acetic acids and their quaternary ammonium salts were synthesized and evaluated for bronchodilator activity by binding assays and pharmacological tests. The in vitro and in vivo functional activity results showed that some quaternary derivatives are potent anticholinergic bronchodilator agents, and 4 compounds ( 14-A, 16-A, 25-A and 28-A) were selected for pharmacotoxicological evaluation. The binding data also indicated that the affinity of these compounds for the tracheal membrane muscarinic receptors is markedly affected by structural modifications of both the cationic head and the acyl moiety of the molecule. In the series of N-alkyl-nor-tropine 2-phenyl-2-cyclohexene-1-carboxylate derivatives, a rather strict limitation of the bulk of the equatorial substituent of tropine nitrogen is required for very high affinity. Conformational analysis and molecular graphics techniques evidenced an influence of the acyl moiety conformation on the affinity of the different tropinic esters, suggesting that the carbonyl oxygen may participate in interaction with the binding site, eliciting a marked increase of potency when it is oriented in a proper conformation with respect to the tropinic nitrogen and the phenyl ring.

Topographic distribution of prostaglandin secretion caused by bradykinin in canine tracheal epithelial cells.

Inflammatory mediators promote the synthesis and secretion of prostaglandin (PG) mediators in airway epithelial cells. In this study, we examined the topographic and kinetic profile of PG secretion in canine tracheal epithelial cells harvested from the tracheal posterior membrane (PM) and those obtained from the immediately anterior cartilage-associated membrane (CM). Primary cultures of tracheal epithelial cells obtained from 23 disease-free dogs were grown to confluence in serum-enriched medium. Cells then were incubated in serum-free medium for 1 h and stimulated with 10(-7) to 10(-5) M bradykinin. Baseline secretion of PGE2 was similar to both PM and CM cells; however, PM cells secreted greater concentrations in both PGI2 (measured as 6-keto-PGF1 alpha) (1,269 +/- 160 versus 775 +/- 91 pg/10(6) cells, P less than 0.01) and PGF2 alpha (436 +/- 54 versus 234 +/- 45 pg/10(6) cells, P less than 0.002) compared with CM cells. Bradykinin (BK) stimulation caused substantial secretion in less than or equal to 20 min of PGE2 and 6-keto-PGF1 alpha from PM but not CM cells: after stimulation with 10(-6) M BK, 6-keto-PGF1 alpha secretion was 348 +/- 74% in PM cells versus 157 +/- 18% of baseline secretion in CM cells (P less than 0.005); PGE2 secretion was 310 +/- 53% in PM cells versus 163 +/- 15% of baseline secretion in CM cells (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Immunopurification and structural analysis of a putative epithelial Cl- channel protein isolated from bovine trachea.

We have purified to homogeneity a 38-kDa protein (called p38) from bovine tracheal epithelium. This protein, when reconstituted into liposomes, mediates stilbene disulfonate-sensitive 125I- conductive uptake. On nonreduced or partially reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this protein associates into a doublet of 62-64 kDa. In some experiments a multimer of 141 kDa was also observed. Rabbit polyclonal anti-P38 antibodies have been produced and used to immunopurify the native transporter. Upon reconstitution of the immunoaffinity-purified protein into liposomes, a 260-fold enhancement of 4,4'-bis(isothiocyano)-2,2'-stilbenedisulfonate and valinomycin-sensitive 125I- uptake was observed as compared to proteoliposomes containing unseparated material. On Western blots of total solubilized tracheal membrane proteins or semipurified fractions, the antibody recognized the 62-64-kDa doublet much better than the original 38-kDa antigen. Similar protein bands were detected in T84 and CFPAC cells as well. However, if apical membrane proteins were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, the antibody recognized major bands at 140 and approximately 240 kDa. Upon partial reduction, immunolabeling of these proteins diminished with the concomitant appearance of the 62-64-kDa doublet. Upon complete reduction, the appearance of 32- and 38-kDa proteins was evident with the disappearance of the 62-64-kDa doublet. We hypothesize that the native Cl-channel is a heteromer containing at least four subunits connected by S-S bridges.

Mucociliary transport with and without the use of a heat and moisture exchanger. An animal study

The importance to mucociliary transport (MCT) and the condition of the mucus of using a heat and moisture exchanger (HME) was investigated. A high tracheotomy was performed on 10 young pigs. The animals were ventilated using a non-breathing system. In five animals an HME was used; the other five were connected directly to the ventilator circuit. After 6 h the trachea was inspected via a flexible bronchoscope. Mucociliary transport velocity was measured using Tc-99-marked macrospheres and a gamma camera. In the control group the tracheal mucus membrane was desiccated at half the distance from the tube tip to the bifurcation. In the HME group the entire trachea was well moistened, but two cases showed large amounts of abnormally thin and foamy secretion. Mucus quality differed significantly between the two groups. Maximum MCT velocities were 8.3 +/- 4.6 and 4.9 +/- 3.0 mm/min for the HME- and the control groups, respectively. The marker had moved 49 +/- 32 and 35 +/- 21 mm during 24 min. Neither of these differences was statistically significant. In the control group there was no transport over desiccated areas. We conclude: 1) the quality of tracheal secretion differed significantly between the two groups, and 2) a heat and moisture exchanger may retain too much water in the airway.

Induction of rhythmic contraction in canine tracheal smooth muscle

Na(+)-K+ ATPase activity of the canine tracheal smooth muscle membrane is responsible for the electrogenic pumping of Na+ and K+ ions. It has been shown that this activity results in muscle relaxation. Based on the results of the current study, we suggest that prolonged electrical stimulation induces increased Na(+)-K+ ATPase activity in isolated tracheal smooth muscle. Tracheal smooth muscle pretreated with prolonged electrical stimulation developed graded mechanical activity when subsequently treated with histamine, serotonin, acetylcholine, or 80 mM K+. This increased isometric tension was interrupted by rhythmic activity, which was elicited by histamine or serotonin but not by acetylcholine or 80 mM K+ stimulation. The spontaneous phasic activity was not inhibited by atropine or propranolol but was totally inhibited by 10(-6) M ouabain. These results suggested that the relaxation phase of rhythmic contraction in response to histamine and serotonin stimulation could be the result of stimulated Na(+)-K+ ATPase activity.

Evidence for vasoactive intestinal peptide receptors in apical membranes from tracheal epithelium

[ 125I]VIP (vasoactive intestinal peptide) bound to apical membranes isolated from the bovine tracheal epithelium with a half maximal inhibition by unlabeled VIP (IC 50) of 0.6 × 10 −9M and binding was reversible. Glucagon did not affect [ 125I]VIP binding to the membranes. [ 125I]VIP was covalently cross-linked to tracheal membrane proteins using disuccinimidyl suberate. SDS-polyacrylamide gel electrophoresis of labeled tracheal membranes revealed one major [ 125I]-receptor complex of Mr = 71,000 to which binding of [ 125I]VIP was inhibited by 10 μM unlabeled VIP. These results are consistent with the presence of a specific, high-affinity receptor for VIP, with a Mr = 71,000, in apical membrane vesicles isolated from the bovine tracheal epithelium.

Sulfate transport in apical membrane vesicles isolated from tracheal epithelium.

Sulfate uptake in apical membrane vesicles isolated from bovine tracheal epithelium is shown to occur into an osmotically sensitive intravesicular space, via a carrier-mediated system. This conclusion is based on three lines of evidence: 1) saturation kinetics; 2) substrate specificity; and 3) inhibition by the anion transport inhibitors SITS and DIDS. The affinity of the transport system is highest in low ionic strength media (apparent Km = 0.13 mM) and decreases in the presence of gluconate (apparent Km = 0.68 mM). Chloride appears to cis-inhibit sulfate uptake and to trans-stimulate sulfate efflux. Cis-inhibition and trans-stimulation studies with a variety of anions indicate that this exchange system may be shared by HCO3-, S2O3(2-), SeO4(2-), and MoO4(2-) but not by H2PO4- or HAsO4(2-). Studies indicate that protons may play two distinct roles in sulfate transport in this system. 1) Their possible modifier role is suggested by the fact that protons affect SO2-4 transport in an uncompetitive manner. 2) The possibility that the proton gradient may act as an energy source for a secondary active transport is indicated by the fact that the imposition of a proton gradient stimulates a transient movement of sulfate in to the tracheal apical membrane vesicle, against its concentration gradient, causing an "overshoot" phenomenon. Our studies show that the carrier-mediated system can function in the absence of chloride. The overshoot observed in the presence of a proton gradient (OH- gradient) indicates that under those conditions the mechanism of transport may be a SO4(2-)-OH- exchange. The fact that chloride cis-inhibits and trans-stimulates SO4(2-) transport indicates that SO2-4 uptake may also occur via a SO4(2-)-Cl- exchange. Studies carried out so far do not enable us to conclude unequivocally whether the tracheal apical membrane system displays two distinct carrier activities (SO4(2-)-Cl-; SO4(2-)-OH-) or one anion exchanger, which like the erythrocyte anion exchanger, may interact with SO4(2-), Cl-, and H+. The fact that the anion transport inhibitors DIDS and SITS inhibit SO4(2-) transport in the presence or absence of chloride suggests that the latter possibility may be the case.

Effect of airway wall flexibility on clearance by simulated cough.

In our previous study, we investigated the relationship between mucus rheology, depth of mucus layer, and clearance by simulated cough. The purpose of the present study was to examine the effect of airway wall flexibility on the clearance of mucuslike gels. Transient airflows similar to cough were generated by both positive and negative pressure, the latter to mimic the dynamic compression that occurs during real cough. As in the previous study, the trachea was modeled as a trough of rectangular cross section with only the bottom lined with the mucus simulant. Clearance was followed by observing the displacement of marker particles. Since cough clearance is intimately related to wave formation in the mucus blanket, we hypothesized that clearance might be impeded if the wave formation occurred simultaneously in the wall and its lining layer. Thus, in one set of experiments the bottom rigid surface of the model trachea was replaced with a frame over which a flexible membrane could be drawn, whereas in the other set the rigid top was replaced by the frame. We also examined the effect of negative-pressure cough in excised canine tracheae, comparing the case where the tracheal membrane was free to deform vs. the case where it was secured. For the rigid-walled model, clearance by positive or negative pressure, with matched flow pattern, was the same. With the mucus simulant lining the flexible bottom surface, clearance increased with increasing membrane flexibility for negative-pressure cough and decreased for positive-pressure cough.(ABSTRACT TRUNCATED AT 250 WORDS)

Transtracheal migration of an intravertebral Steinmann pin to the left bronchus

Foreign body aspiration only rarely occurs by migration into the lung from other sites in the body. This report describes the migration of an intracervical Steinmann pin through the posterior tracheal membrane into the left bronchus and its successful removal via the rigid bronchoscope.

The latissimus dorsi muscle flap is useful for the repair of tracheal defects--an experimental study.

An experiment was designed to evaluate the limitations of the muscle flap method in treating various locations and sizes of tracheal defects. Five groups of dogs with several types of tracheal defects were prepared. Each defect was covered by the latissimus dorsi muscle flap. Defects of the tracheal membrane (posterior one-third of the tracheal wall) were successfully repaired by the muscle flap without stenosis, even if they extended to 10 rings in length or were situated at the carina. Defects in the posterior one-half of the trachea, up to 5 rings long, were repaired, with minimal stenosis. Defects of the posterior two-thirds, or anterior one-third of the trachea, resulted in marked stenosis following muscle flap repair. Tracheal movement as seen in the saver sheath type of tracheomalasia was observed when anterior support of the trachea was lost. On the other hand, the movement seen in the crescent type of tracheomalasia was observed when posterior support of the trachea was lost. We concluded that a defect of less than the posterior one-half of the trachea can be repaired by muscle flap, without inducing respiratory insufficiency. Therefore, muscle flap coverage for tracheal defects should be a useful technique in the combined resection of the tracheobronchial tree in cases of esophageal cancer.

全周側方照射型ＹＡＧレーザーファイバーによる気管内膜破壊の研究 水酸化アパタイト人工気管の開発

Sintered hydroxyapatite tube implantation following YAG laser irradiation to the tracheal membrane was evaluated.Pathological changes in the tracheal membrane produced by YAG laser irradiation, which reduces mucus production and accordingly prevents accumulation between the membrane and the prosthesis, were studied in 18 mongrel dogs. A lateral irradiation probe was inserted into the trachea and the inner membrane of the trachea was irradiated circumferentially. The temperature of the membrane was monitored with a thermospot sensor throughout this procedure. The irradiated tissues were examined pathologically with reference to the specific temperature of the membranes. The membranous portion of the trachea showed a higher temperature than the cartilagenous portion with the same laser energy density. Histologically, the grade of destruction was divided into five grades ranging from desquamation to perforation.The most adequate degree of membrane destruction was confirmed as grade 3 which showed shallow localized ulceration reaching to the submucosal glands and the temperature of the membranous portion was approximately 60°C.The results obtained from the present study indicated that a hydroxyapatite prosthesis could adhere to the inner wall of the trachea most firmly with pretreatment by YAG laser irradiation to get 60°C of the tissue temperature.

YAGレーザー気管粘膜照射による気管内膜粘膜層破壊

YAGレーザー気管粘膜照射による気管内膜粘膜層破壊

Solubilization and reconstitution of a chloride transporter from tracheal apical membrane.

Electrogenic Cl- transport in bovine tracheal membrane vesicles was studied by measuring the kinetics of Cl- efflux. Efflux from KCl-loaded vesicles was assayed as the appearance of Cl- in the medium using an anion-specific electrode. Maximal sensitivity was obtained by passage of the vesicles through a Sephadex column immediately before the assay to remove external Cl-. Two components of Cl- transport could be detected. Vesicles exhibited a characteristic spontaneous Cl- efflux that appears to be limited by the cation permeability of the membrane. Addition of the K+-conducting ionophore to increase selectively the permeability to K+ resulted in a stimulation of Cl- efflux. The electrically neutral ionophore nigericin caused only a slight transient increase in Cl- efflux. The valinomycin-dependent flux may be assumed to represent flux through a parallel conductive pathway. Valinomycin-dependent electrogenic Cl- flux was 240 +/- 36 nmol X mg protein-1 X min-1 (n = 4). Soluble extracts obtained with the nonionic detergent octyl glucoside were reconstituted by the freeze-thaw procedure. The reconstituted vesicles exhibited a number of similarities to the native vesicles. Efflux was maximally stimulated with the ionophore valinomycin in the presence of an outwardly directed K+ gradient. Nigericin had no effect on efflux, suggesting the reconstituted transporter is a conductive rather than exchange mechanism. The initial rate of the valinomycin-dependent component was 896 +/- 63 nmol X mg protein-1 X min-1 (n = 5), representing approximately a fourfold increase in specific activity compared with the native membrane vesicle.

Relationship of the Donnan potential to the transmembrane pH gradient in tracheal apical membrane vesicles.

Experiments were performed to determine the factors which contribute to the transmembrane pH gradient (delta pH) and the potential gradient (delta psi) in apical plasma membrane vesicles isolated from bovine tracheal epithelium. As indicated by the accumulation of 14C-methylamine, the vesicles maintained a delta pH (inside acidic) which was dependent upon the external pH. The delta pH was also proportional to the ionic strength of the suspending medium, suggesting that the H+ distribution was dictated by a Donnan potential. Measurements of the distribution of 86Rb+ demonstrated an electrical potential gradient across the vesicle membrane, inside negative which was proportional to the medium ionic strength. delta pH changed in parallel with delta psi in response to a variety of imposed conditions. These results are compatible with the existence of a H+ conductance in the vesicle membrane. Thus the endogenous electrical and proton gradients may be manipulated and used as a general experimental tool to complement kinetic analysis in investigations of transport mechanisms using isolated vesicle preparations.

粘液線毛輸送系の超微形態

Fine structures of cilia of airways in rats and mice, including the microtubular organization, crown, basal body and rootlet, were outlined. Fixation from behind the walls of airways reserved the surface film covering the mucous membrane and the trachea was shown to be covered with two layers; all cilia were underneath a thin top layer of fine fibrillar material and the tips were sticked into the underside. On the other hand, the bottom layer where the main body of a cilium was located, was lucent. In small airways, all cilia were embedded in a single layer of fine granular material. In addition, changes in tracheal mucous membrane in rats which received a single dose of porcine pancreatic elastase intratracheally were reported. The principal finding seen 12 hours after elastase instillation was the nonciliated cells covering almost totally the surface of the trachea. Ciliogenesis was a very frequent finding and distinct increment of ciliated cells was seen by 72 hours. These findings were compared with those seen in the mucus membrane of the bronchial walls in patients with chronic bronchitis.

Cl − transport in apical plasma membrane vesicles isolated from bovine tracheal epithelium

The Cl − transport properties of the luminal border of bovine tracheal epithelium have been investigated using a highly purified preparation of apical plasma membrane vesicles. Transport of Cl − into an intravesicular space was demonstrated by (1) a linear inverse correlation between Cl − uptake and medium osmolarity and (2) complete release of accumulated Cl − by treatment with detergent. The rate of Cl − uptake was highly temperature-sensitive and was enhanced by exchange diffusion, providing evidence for a carrier-mediated transport mechanism. Transport of Cl − was not affected by the ‘loop’ diuretic bumetanide or by the stilbene-derivative anion-exchange inhibitors SITS (4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid) and DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid). In the presence of the impermeant cation, tetramethylammonium (TMA +), uptake of Cl − was minimal; transport was stimulated equally by the substitution of either K + or Na + for TMA +. Valinomycin in the presence of K + enhanced further Cl − uptake, while amiloride reduced Na +-stimulated Cl − uptake towards the minimal level observed with TMA +. These results suggest the following conclusions: (1) the tracheal vesicle membrane has a finite permeability to both Na + and K +; (2) the membrane permeability to the medium counterion determines the rate of Cl − uptake; (3) Cl − transport is not specifically coupled with either Na + or K +; and, finally (4) Cl − crosses the tracheal luminal membrane via an electrogenic transport mechanism.