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We describe a stepwise-development thin-layer-chromatographic technique for separating phosphatidylglycerol and phosphatidylinositol in samples of amniotic fluid. Plates are prepared from silica gel G slurried in a 50 g/L ammonium sulfate solution. Phospholipid phosphorus determination is not needed. Phospholipid phosphorus determination is not needed. Phosphatidylglycerol and phosphatidylinositol, measured densitometrically, are expressed as ratios to the sphingomyelin present. These ratios are correlated with the lecithin/sphingomyelin ratio and age of gestation in normal and abnormal pregnancies.

In the present investigation, a histochemical study of lipids have been made in the trachea and lungs of common ground squirrels exposed to three chief air-pollutants. Significant changes thus observed in pulmonary lipids have been correlated with altered mechanical properties of the lung and morphology. Supplementary biochemical findings made on total lipids indicate that CO ranks at the top effecting lipid contents of both trachea and lungs than are the SO2 and NO2 respectively. Physiological mechanism of damage has also been analysed.

We measured tracheal fluid production rate and pulmonary surfactant flux in 12 sets of twin fetal lambs of 102 to 135 days' gestation. In nine of these sets we also measured surfactant flux and the concentration of saturated lecithin in the lungs before and during dexamethasone infusion into one of the twins. The average tracheal fluid flow rate was 3.25 ml/kg/hr (SD 1.6) and, relative to body weight, did not change between 103 and 135 days' gestation or during the infusion of dexamethasone. In untreated fetuses, surfactant was detected between 108 and 130 days and its flux gradually increased but remained less than 150mug/kg/hr at 135 days' gestation; the amount of disaturated lecithin in the lung relative to body weight increased 13-fold from 108 to 134 days. From these data we calculated that the minimal rate of synthesis of disaturated lecithin was increased about 4-fold by dexamethasone from 108 to 120 days; this enhancement fell to about 1.8-fold by 135 days. The rate of secretion of disaturated lecithin, as estimated by surfactant flux in tracheal fluid, was increased by dexamethasone throughout the interval of the study.

The percentage area of the bronchial glands taking up the Alcian blue (pH 2-6) stain for acid mucin was obtained from sections from the trachea and from each generation down the inferior lingular axial bronchial pathway in five non-smokers and five cigarette smokers. The mean percentage value for the smokers (59-5%) was significantly less than for the non-smokers (74-4%). The percentage increased between successive generations from the trachea by an average of 6-5. This gradient did not differ significantly between the smoking and non-smoking groups. It was not possible to attribute these differences to any specific mechanism because many interacting variables were involved which could not be measured. Nevertheless the percentage mucin retention or storage provides an introduction to consideration of these variables and their response to cigarette smoke.

Chloride channels mediate absorption and secretion of fluid in epithelia, and the regulation of these channels is now known to be defective in cystic fibrosis. Indanyl-oxyacetic acid 94 (IAA-94) is a high-affinity ligand for the chloride channel, and an affinity resin based on that structure was developed. Solubilized proteins from kidney and trachea membranes were applied to the affinity matrix, and four proteins with apparent molecular masses of 97, 64, 40, and 27 kilodaltons were eluted from the column by excess IAA-94. A potential-dependent 36Cl- uptake was observed after reconstituting these proteins into liposomes. Three types of chloride channels with single-channel conductances of 26, 100, and 400 picosiemens were observed after fusion of these liposomes with planar lipid bilayers. Similar types of chloride channels have been observed in epithelia.

NaCl myosin was prepared from the annular smooth muscles of human bronchus. About 7 mg of gel filtered myosin was gained from 8 g minced tracheal muscle of the younger subject. The yield from the older (74-year old) subject was only 30% of that from the younger subject, even though the starting material was more (12 g minced tissue). Tracheal myosin contains P lipid in considerable amount; P lipids account for some 28% of the total phosphate content of the myosin, and even more (50-55%) in the case of the older subject. The preparation could be phosphorylated only in the presence of CAMP and PGF2 alpha, respectively. Cu2+ treatment liberated less phosphate when compared with myosin preparations from other smooth muscles; however, the majority of the phosphate bonds underwent hydrolysis upon the effect of KOH. The reactions specific for amino acids, and also other observations allow the conclusion that the majority of covalently bound phosphate is present in an ester-type bond. Lysine-vasopressin, and also diethylpyrocarbonate successfully protect the P content of myosin from the hydrolysis inherent to incubation.

A procedure is described for the direct collection of bronchial or tracheal fluid samples on to paper discs. Using the procedure, the pharmacokinetics of ceftazidime in rabbit bronchial and tracheal fluids were compared with those in the respective wall tissue samples and in lung tissue. Concentrations appearing in lung tissue were approximately half those seen in bronchial or tracheal fluid or bronchial and tracheal wall tissue. Concentrations in these latter compartments were, in turn, four- to six-fold lower at all times than simultaneously-measured serum levels. The shape of the concentration/time curves were similar for all compartments sampled. The half life values were 63 to 64 min for respiratory tract concentrations and 59 min for serum levels. Percentage penetration from serum into the various compartments was 20.8% for bronchial fluid, 19.9% for tracheal fluid, 22.4% for bronchial wall, 20.0% for tracheal wall and 11.3% for lung tissue.

Galanin, a newly discovered peptide isolated from porcine intestine, is known to cause contraction in rat smooth muscle preparations and to induce hyperglycaemia in dogs. By the use of radioimmunoassay and immunohistochemical techniques the concentration and distribution of galanin immunoreactivity were determined in several areas of the respiratory tract of five dogs, five guinea pigs, five rats, and two pigs. Antibodies were raised in rabbits to whole unconjugated natural porcine galanin. The highest galanin concentrations were found in the bronchus and the trachea of the dog, guinea pig, rat (2 pmol/g in each case), and pig (less than 1 pmol/g). The lowest galanin concentrations were found in the lung parenchyma. Gel chromatographic analysis in the pig showed one molecular form of galanin coeluting with the porcine galanin standard. By means of the indirect immunofluorescence technique on sections of tissues fixed in benzoquinone solution, galanin was found to be confined to nerve fibres in different regions of the respiratory tract. In the nasal mucosa of the pig nerve fibres containing galanin were distributed around seromucous glands and blood vessels and beneath the epithelium. In the trachea, bronchus, and major intrapulmonary airways of the pig, dog, and guinea pig galanin immunoreactive fibres were detected predominantly in smooth muscle, as well as around seromucous glands and in the adventitia of blood vessels. Rarely, galanin immunoreactive nerve fibres were found in the lung parenchyma. A few galanin immunoreactive ganglion cells also containing vasoactive intestinal polypeptide were found in the adventitia of the tracheobronchial wall of the pig and dog. The distribution of galanin suggests that it may have some influence on airway, vascular, and secretory functions in the mammalian respiratory tract.

To elucidate the biochemical basis of airway hyperreactivity, we studied the relationships between in vivo airway sensitivity of guinea pigs to histamine and their tracheal beta-adrenergic binding sites, Ca++- and (Na+-K+)-ATPase activities, and composition of phospholipids. The relationships between tracheal and plasma phospholipids were also examined. beta-Adrenergic receptor binding with 3H-dihydroalprenolol in tracheal tissue showed an inverse relationship with in vivo airway sensitivity to histamine. Among the phospholipids, tracheal phosphatidyl ethanolamine content varied inversely with in vivo airway sensitivity, whereas tracheal and plasma lysophosphatidyl choline contents showed a direct correlation with airway sensitivity. A significant direct correlation was also observed between tracheal and plasma lysophosphatidyl choline levels. Both Ca++-ATPase and (Na+-K+)-ATPase activities increased with increasing airway sensitivity. These enzymes showed inverse correlations with phosphatidyl ethanolamine content and direct correlations with lysophosphatidyl choline content. Our data suggest that increased lysophosphatidyl choline may cause various biochemical changes associated with airway hyperreactivity.

The effect of aspiration of blood on pulmonary host defenses was studied in the rat. Sham and experimental rats had 0.2 ml of saline or blood/100 g body weight injected into their tracheas. One or 24 hours after aspiration rats were challenged with aerosolized, radiolabeled ((32)P), S. aureus. Fourteen hours after bacterial challenge, lungs were removed and intrapulmonary bacterial inactivation was quantified. Significant impairment of bacterial inactivation occurred at both 15 and 38 hours after aspiration of blood, but not after saline. The pulmonary consolidation after aspiration of blood was focal in nature. The lung weight increased but fractional water content decreased. Arterial pH, pCO(2), or pO(2) were unaffected by aspiration of blood. The number and viability of macrophages recovered by lavage were similar in control, sham and experimental groups. If similar impairment in pulmonary host defenses occurred in man following aspiration of blood, the patient with aspiration of blood would have an increased susceptibility to bacterial infection.

Tracheas from 15 toy breed dogs with normal tracheas and 6 dogs with collapsed tracheas were examined histologically and histochemically. Tracheal cartilage was analyzed for chondroitin sulfate by means of alcian blue (CEC method) and for calcium with alizarin red S. Cartilage arcs from dogs with collapsed tracheas had areas that were apparently hypocellular, and some had other areas that appeared like fibrocartilage or fibrous tissue. Histochemically, collapsed tracheal cartilage had less chondroitin sulfate and calcium than did normal tracheal cartilage. Cartilage arcs from the collapsed tracheas were not uniformly affected to the same degree, and parts of a given tracheal arc appeared normal, whereas other parts had an abnormal histologic appearance.

The expectorant effect of brovanexine (BvX) was investigated histologically and histochemically using isolated canine trachea. Following BvX treatment, the number of goblet cells (GC) stained positively with the combination procedure of alcian blue at pH 2.5 or pH 1.0 and periodic acid-Schiff was unaffected. The number of GC which stained blue and purple (stain index B & P) was reduced, while the number of GC that stained red (stain index R) was increased. The thickness of the acini of submucosal glands (SG) markedly decreased with 10(-5) and 10(-4)M BvX, and the ratio of the acinar inner diameter to the tracheal wall thickness significantly increased with 10(-4)M BvX. Stain index B & P of glandular cells decreased with 10(-5) and 10(-4)M BvX, which was accompanied by a marked increase in the stain index R. BvX treatment caused increases in total saccharide and protein concentrations in the incubation fluid, while N-acetylhexosamine slightly decreased. Both the BvX-induced histological changes in glandular cells and changes in concentrations of macromolecular components in the incubation fluid were much the same degree as those induced by bromhexine (Bh). The BvX-induced histochemical changes in secretory cells, however, were slighter than those induced by Bh. These findings suggest that BvX is an expectorant possessing both the secretagogic action selectively on SG and a mucolytic action toward acid glycoproteins in granules of secretory cells.

Changes in the expression of heavy chains of myosin during development determine the functional characteristics of striated muscles. The distribution of heavy-chain isoforms of smooth-muscle myosin was determined in the airways of adult and infant humans to see whether it might underlie the hyperreactivity of human airways. The protein bands corresponding to myosin were separated using SDS/polyacrylamide-gel electrophoresis (4% gels) and identified by immunoblotting using both monoclonal and polyclonal antibodies against smooth-muscle myosin and non-muscle myosin. The relative proportion of each heavy chain stained by Coomassie Blue was measured by densitometric scanning. Three major bands corresponding to myosin heavy-chain isoforms were found; the two slower migrating bands (MHC1 and MHC2) were smooth-muscle myosin, and the third band was non-muscle myosin. The MHC1/MHC2 ratio was 0.69:1 in adult bronchus, and in infant bronchus and trachea. This contrasted with the airway smooth muscle in pigs, which was run concurrently, where the smooth-muscle heavy-chain ratio changed with development [Mohammad & Sparrow (1988) FEBS Lett. 228, 109-112]. The non-muscle myosin heavy chain comprised 63% of the smooth-muscle myosin. In both adult and infant lungs an additional putative myosin heavy chain which migrated slightly more rapidly than non-muscle myosin heavy chain was identified using the monoclonal smooth-muscle myosin antibody BF 48. This was unique to the human species.

During the regeneration of the mucociliary lining of the respiratory airways, many cells differentiate into ciliated cells. Early stages of ciliogenesis in these cells is characterized morphologically by appearance of MTOC, filosomes and centrioles throughout the apical cytoplasm. Tracheas removed during the period of ciliated cell differentiation which occurs 50-60 hr after minor mechanical injury were paraformaldehyde fixed and specific affinity sites for antitropomyosin and antimyosin antibodies were demonstrated by an indirect immunoperoxidase technique. The epithelium, after development of the osmiophilic reaction product, was embedded in epoxy and observed unstained with an electron microscope. Both antibodies had similar and specific binding sites in filosomes, MTOCs, microfilaments and on the microtubule triplets and foot processes of centrioles. Such localization suggests that these mechanochemical proteins may in addition to microfilament stabilization and contraction serve a specialized function in ciliogenesis.

Ciliated tracheal epithelia cell cultures were investigated immunocytochemically with anti-tubulin and colloidal gold. When rabbit tracheal cultures were fixed in paraformaldehyde, treated with acetone, anti-tubulin and a second antibody coupled to FITC, fluorescence was associated with cytoskeletal and axonemal microtubules. Cilia covering the apical surface of the ciliated tracheal cells fluoresced very brightly thus facilitating identification of this cell type. Electron microscopy of tracheal cultures fixed as above, treated with Triton-X 100 and incubated in anti-tubulin and protein A coupled to colloidal gold resulted in the highly specific localization of tubulin in ciliary axonemes and basal bodies. Omission of primary or secondary antibody resulted in extremely low levels of fluorescence while no colloidal gold particles could be detected in cultures at the electron microscopy level when rabbit anti-tubulin was omitted.

Four monoclonal antibodies raised to alpha subunit of pig brain tubulin (TU-01, TU-02, TU-03, TU-04) were used to study immunohistochemical heterogeneity of alpha tubulin in human epithelia. Selective reactivity was detected in the skin and trachea/bronchi, whereas all other epithelia investigated reacted uniformly with all four monoclonal antibodies. In the skin TU-01 reacted very strongly with all layers except the basal layer; TU-02 reacted strongly with granular layer and was unreactive or only weakly reactive with others; TU-03 reacted very strongly with basal layer and weakly to moderately with superficial layers; TU-04 reacted strongly with the granular layer of epidermis and was unreactive with other layers. In the trachea and major bronchi TU-01 reacted with the entire epithelial layer; TU-02 reacted only with superficial layer; TU-03 reacted with superficial and basal layer; TU-04 reacted only with superficial layer. Different staining patterns obtained with these four monoclonal antibodies indicate that there is immunohistochemical heterogeneity of alpha tubulin in some but not all normal human epithelia.

Connective tissue macromolecules, glycosaminoglycans, glycoproteins, collagen, and elastin were isolated from different parts of the respiratory system and characterized. The materials included bronchiolar tissue, gas-exchange tissue, lung pleura, and tracheal mucosa. The similarity of the macromolecular composition of lung pleura and tracheal mucosa suggests a common cellular component in these structures. The high concentration of GAG and collagen in bronchiolar tissue is consistent with the cartilagenous nature of this tissue. Particularly interesting is the high content of heparin in all pulmonary structures, a relatively greater content of hyaluronic acid in gas-exchange tissue, and a high content of heparan sulfate and dermatan sulfate in vascular tissues. Elastin also occurs as a major fibrous structure. Although the biologic role of these connective tissue macromolecules has not been established, certain functional relationships are inferred.

The efficiency and accuracy of four different methods of tissue preparation for the recovery of dotriacontane-16,17-14C (DOT-16,17-14C) from rat respiratory tissue were compared by means of liquid scintillation counting. A DOT-16,17-14C standard solution was placed on the respiratory epithelium of the nasal apex, larynx, trachea and main bronchi. Combustion of the lyophilized organs in a Packard sample oxidizer revealed the highest recovery and accuracy (93--100%), sample oxidizing by combustion without lyophilization the lowest recovery and accuracy (73--92%). Tissue solubilization by the commercially available tissue solubilizer TS-1 revealed a better recovery and accuracy (89--95%) than tissue maceration by boiling in methanolic KOH and toluene extraction (74--91%). Depending on the laboratory equipment, lyophilization or tissue solubilization is preferred; maceration and toluene extraction as well as combustion without freeze-drying are disregarded for further investigations because of the cumbersome procedure as well as the low recovery and accuracy, respectively.

Monoclonal antibodies, 17B1 and 17Q2, which are specific for large molecular weight mucous glycoproteins of airway epithelium, have been used to develop an ELISA method to quantitate the tracheal mucins of humans and rhesus monkeys. The assay is a double-sandwich system that does not depend on either the binding of mucous antigens to the microtiter plate or the use of a second antibody. The assay protocol includes (1) coating the microtiter well with purified IgG of 17B1 or 17Q2, (2) incubating the wells with mucous samples, (3) binding of alkaline phosphatase-conjugated IgG to the wells, and (4) developing the color with phosphate substrate. This ELISA method is very sensitive for human and rhesus monkey tracheal mucins. Quantitation is not affected by the presence of various proteoglycans (keratan sulfate, hyaluronate, heparin, heparan sulfate, and chondroitin sulfate). However, the quantitation is affected by the treatment of antigen with periodic acid and endo-beta-galactosidase. Other enzymes (e.g., neuraminidase, hyaluronidase, chondroitinase, heparitinase, heparinase, fucosidase, keratanase) have no effect on the antigenicity of substrate. The quantitation is linear, with a concentration from 0.2 to 4 ng protein/sample. The ELISA method developed in this study should be useful for quantitating the mucin content of various biologic fluids, such as sputum, bronchoalveolar lavage, and media from cultures following various pharmacologic and physiologic manipulations.

Endogenously fluorescent, singly occurring amine-containing cells in tracheal epithelium were examined in 3-, 10-, and 28-day-old rabbits. These cells are pyramidal in shape with the apex projected toward the tracheal lumen. The cytoplasm exhibits a yellow fluorescence which is predominantly supranuclear. Occasional, infranuclear, fluorescent cytoplasmic processes project from the cells. The numbers of fluorescent cells per unit length of trachea increase with age. Acute exposure of 10-day-old rabbits to 13% O2 decreases the number of detectable fluorescent cells in the tracheal compared to controls exposed to room air. Similarly, exposure to 750 ppm carbon monoxide decreases the number of fluorescent epithelial cells appearing in tracheas of 10- and 28-day-old rabbits. These results suggest that the amine-containing epithelial cells of the trachea respond to tissue hypoxia and that decreased airway pO2 is not necessary to elicit a response.

A simple post-embedding technique for the electron microscopical detection of lectin-binding sites using thin sections of tissues embedded in the resin LR White is described. With this technique, no prior etching of the sections is necessary. The cellular fine structure is well preserved and permits close correlation of the labelling to distinct cellular compartments. After mild aldehyde fixation (4% formaldehyde and 0.5% glutaraldehyde for 30 min), enterocyte brush border, vesicles and lysosomes as well as goblet cell Golgi apparatus and mucin are intensely stained after 30-60 min. The hydrophilia and penetrability of LR White is shown by the formation of oxidized diaminobenzidine reaction product arising from horseradish peroxidase-conjugated lectins. The precipitate not only covers the surface of the sections but is also formed within the resin, as is revealed on cross-sections through thin and semithin sections. The addition of 0.2 M solutions of the appropriate inhibitory sugars prevented staining, which indicates a specific binding. Examples are given of the binding of gold-, ferritin- and peroxidase-conjugated lectins for the purpose of detecting glycoconjugates in various intracellular compartments.

A sensitive double antibody and heterologous enzyme immunoassay for mabuterol was established. For competitive reactions, antibody raised against diazotized mabuterol-human serum albumin was incubated with a mixture of diazotized mabuterol analog (RC-1) labeled with beta-D-galactosidase and standard or sample. Free and antibody-bound enzyme hapten were separated using anti-rabbit IgG immobilized on polystyrene balls. Activity of the enzyme on the solid phase was fluorometrically determined. The present immunoassay allows detection of 0.5 to 100 pg/tube of mabuterol. Pharmacokinetic behavior of this agent in human plasma and urine was studied after a single oral administration (50 micrograms). The maximum level was achieved after 2-3 hrs with approximately 280 pg mabuterol /ml of plasma and the half life of mabuterol was estimated to be 19.5 hrs. Cumulative amount of mabuterol in the first 72 hrs urine was 64.3 +/- 13.2% of the administered dose.

Three homogeneous glycoproteins were isolated from reduced and S-carboxy-methylated canine tracheal pouch mucus by gel filtration and ion-exchange chromatography. Initial fractionation was carried out on Sephadex G-200; chromatography of the excluded Sephadex G-200 fraction on Bio-Gel A-15 m yielded two high molecular weight glycoprotein fractions. Following rechromatography on the same column, the main fraction behaved as an electrophoretically homogeneous high molecular weight (581 600) glycoprotein, with a high carbohydrate content (80%) and a single amino-terminal amino acid (arginine). Ion-exchange chromatography (DEAE-cellulose) of the included Sephadex G-200 fraction yielded two electrophoretically homogeneous glycoproteins of lower molecular weight (20 800 and 24 600, respectively). A single amino-terminal amino acid, glycine and alanine, respectively, was detected for each glycoprotein. Chemical analysis of these three glycoproteins revealed the presence of fucose, galactose, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid and sulfate monoester. The high molecular weight glycoprotein had a higher hexose, sialic acid and sulfate content, per mg of protein, than the low molecular weight glycoproteins. The results of the alkaline borohydride treatment indicated that the majority of the carbohydrate chains of these glycoproteins are linked to the protein core through O-glycosidic bonds involving N-acetylgalactosamine and serine or threonine.

H1-receptor reserves in guinea-pig left atria, trachea and pig coronary arteries were calculated by the use of phenoxybenzamine (Pba), a beta-haloalkylamine that irreversibly blocks the H1-receptor response to histamine or 2-(2-pyridyl)-ethylamine (PEA). Equieffective concentrations of the H1-agonist in the absence (A) and presence (A') of Pba were evaluated from concentration-response curves. By plotting the reciprocal values 1/A versus 1/A' the amount of H1-receptors not occupied by the agonist was calculated. The size of the H1-receptor reserve could be estimated by comparison of the receptor occupation with the corresponding effect. Furthermore, the dissociation constants for histamine, PEA, Pba and the pD'2-values for Pba were determined for the different tissues. 5% and 6% H1-receptor occupation is necessary to achieve a half maximal contraction of the trachea with the agonists histamine and PEA, respectively. Only 0.5% H1-receptor occupation is needed for the half maximal positive inotropic effect of histamine in left atria, while 5.5% of the H1-receptors have to be occupied using PEA as an agonist in this tissue. In the coronary artery of the pig 50% of the maximal contraction can be achieved by stimulation of 15.1% of the H1-receptors with histamine.

The possibility of the penetration of pollen grains into the tracheobronchial tree is still a controversial question. To investigate this phenomenon further two different approaches were conducted: 1) Acetolysis (acidic degradation of organic constituents) of mucosal samples from different levels of the pig respiratory tract showed that specific (plane tree) inhaled pollens were not found beyond the main bronchi. 2) In contrast, a noticeable number of pollens were present in acetolyzed residues of lung surgical samples; this was shown to be due to contamination. In the first approach this problem was avoided; however, the analysis of surgical samples could be a valuable technique if suitable controls were included.

We characterized the chemical composition of mucins secreted by ferret tracheal explants and the activities of key mucin glycosyltransferases in ferret tracheal epithelium during a period of rapid postnatal maturation of the mucin-secreting structures. Ferret tracheal explants secrete three major groups of high molecular weight glycoconjugates: (1) those susceptible to bovine testicular hyaluronidase; (2) those resistant to hyaluronidase and exhibiting high density (p greater than or equal to 1.60 g/mL); and (3) those resistant to hyaluronidase and exhibiting low density (1.45 less than or equal to p less than 1.60 g/mL). The hyaluronidase-resistant, low-density glycoconjugates have typical mucin properties and constitute 36% of total glycoconjugates released in newborns but only 8% in adult ferrets. Mucin secretory rate per unit surface area of trachea progressively decreases with age. Mucin amino acid and total carbohydrate contents do not vary; however, the sialic acid content increases, and fucose content as well as blood group A activity of the mucins decreases with age. Four glycosyltransferases involved in mucin biosynthesis [Gal beta 3GalNAc:(GlcNAc-GalNAc)beta 6 N-acetylglucosaminyl-, GalNAc:beta 3 galactosyl-, Gal:alpha 2 fucosyl-, and GalNAc alpha 2----6 neuraminyltransferase] are present in tracheal epithelium of ferrets at all ages. Activities of all but the neuraminyltransferase decrease with age. The relatively greater neuraminyltransferase activity is consistent with increased incorporation of sialic acid into secreted mucins over the same age span. Conversely, diminution of fucosyltransferase relative to galactosyltransferase activity may contribute to the lower fucose content and lower blood group A activity of mucins secreted by mature ferret tracheas.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide tyrosine (NPY), a newly discovered peptide known to modulate blood vessel diameter and smooth muscle tone, has been found in many mammalian organs. Its distribution is similar to that of sympathetic nerve fibres and NPY immunoreactivity has been found in noradrenergic ganglion cells. In a study of the respiratory tract of four mammalian species--man, cat, guinea pig, and rat--NPY immunoreactivity has been localised to nerve fibres. NPY immunoreactive nerve fibres were found in the adventitia of blood vessels and in the airway smooth muscle. Its distribution was strikingly similar to that of sympathetic nerve fibres as demonstrated by dopamine-beta-hydroxylase antibodies. The mean (SD) concentrations of NPY in the guinea pig respiratory tract, as determined by radioimmunoassay of tissue extracts, were: upper trachea 3.3 (0.7), lower trachea 2.0 (0.5), and major bronchus 3.5 (1.1) pmol/g. During developmental studies in man NPY immunoreactive nerve fibres were first observed at 20 weeks' gestation in the trachea, and fibres gradually extended down into the intrapulmonary airways after birth. NPY immunoreactive nerve fibres have a distribution and developmental pattern similar to that of sympathetic nerve fibres in the respiratory tract. The finding of NPY immunoreactivity in nerve fibres in the mammalian respiratory tract adds to the growing number of peptides having potent biological actions found in this organ, and shows that the lung possesses a rich peptidergic system, which may influence pulmonary function.

To determine the molecular composition of the components of basal bodies and the interbasal body apparatus of ciliated cells in rat tracheal epithelium, we used rabbit anti-actin, anti-alpha-actinin, anti-tropomyosin, and anti-myosin as primary antisera applied to the tissue in an indirect immunoperoxidase technique. The antisera was proven to be monospecific by elution of antibody after affinity chromatography. Sheep anti-rabbit immunoglobulin Fab fragments coupled to peroxidase were used for ultrastructural localization of the bound rabbit antibody. Antibodies against alpha-actinin were demonstrated around peripheral microtubules of cilia and linking these microtubules to central doublet and plasma membrane. Alpha-actinin was also shown in the basal foot processes. Anti-actin antibodies were associated with microtubules of the cilium and basal bodies, except in the region of the ciliary necklace. The antibodies directed against actin also had affinity for rootlets, basal foot processes, and communications between basal bodies and foot processes. Both anti-myosin and anti-tropomyosin antibodies were localized to part of the region of the constriction of the cilium, to the central basal density and the outer surfaces of basal body microtubules, and to the basal foot processes together with their communications to the basal body. The data suggest active contractile function of basal bodies.

In order to study the accumulation of collagen during postnatal development in the major structures of the lung, trachea, bronchi, pulmonary vasculature, and parenchyma were isolated from the lungs of 1-week, 2-month, and 6-month-old rabbits. The concentrations of DNA, protein, and collagen in parenchyma were similar to those for whole lung. DNA per day weight for all structures declined with age. The DNA concentration of trachea at 6 months was only one-tenth that of parenchyma. Protein per dry weight was constant in parenchyma but dropped by half in trachea and nearly that in bronchi. Collagen per dry weight increased significantly between 1 week and 6 months in all structures. The collagen concentrations in vessels, bronchi, and trachea are similar to each other and three to five times that of parenchyma. The total amount of collagen in bronchi, trachea and parenchyma increased approximately 12-, 50-, and 90-fold, respectively, between 1 week and 6 months of age.

The lungs of eight individuals were investigated for 27 elements by neutron activation analysis and atomic absorption spectrometry. Thirteen segments of each lung pair were analyzed, together with hilar lymph nodes. The trace element distribution in the lungs and the inter-individual concentration differences reveal the existence of two groups of elements. A similar situation is observed in the lymph nodes. The elements Br, Cs, Cu, K, Na, Rb, Se and Zn are relatively homogeneously distributed over a lung pair and show little inter-individual concentration differences. On the other hand, Cd, Co, Cr, Pb, Sb, Sc and V are very inhomogeneosly distributed. The inhomogeneity and the concentrations increase with age. The existence of a concentration gradient within the lung and of relatively higher levels in the lymph nodes point to an enrichment in the lung tissue by inhaled atmospheric contaminants.

To estimate the role of vitamin A on ciliated cells we investigated whether ciliated cells undergo any alteration during vitamin A deficiency. The epithelia examined include the ciliated cells of the respiratory tract and the ciliated sensory cells of the inner ear, the tongue, and the olfactory cells. This part of the paper will describe the ciliated epithelium of the tracheobronchial tract and its relation to vitamin A status. During vitamin A deficiency a partial loss of ciliae can be observed before any squamous metaplasia (which usually occurs during longer lasting vitamin A deficiency) develops. The scanning electron microscopic data illustrate the altered surface of the epithelium during vitamin A deficiency better than transmission electron microscopy.

The surfactant phospholipids in the tracheal effluent of eight pairs of preterm twins were followed, serially. In four pairs only one of the twins had RDS; in the other four pairs both twins had RDS. Twins with and without RDS had markedly different surfactant phospholipids. Phosphatidylglycerol was absent in all twins with RDS. L/S ratios in tracheal effluent increased after birth in infants with and without RDS. With recovery from RDS, phosphatidylinositol increased. The study helps confirm previous studies of the necessity of the acidic phospholipids, PI and PG, in stabilizing lecithin, as well as that surfactant composition can be independent of gestational age.

The microviscosity of tracheal aspirates from newborn children with respiratory problems was investigated using fluorescence polarization. The pulmonary status of 100 newborns with hyaline membrane disease (HMD) and a reference group (n = 45) was assessed daily throughout of intubation. Two main changes in phospholipid microviscosity (eta) were identified. A decrease in eta associated with a favourable clinical outcome. A dramatic increase in eta with an unfavourable outcome in pulmonary function. These changes correlated with clinical observations, but the trends in microviscosity seemed to appear before any related clinical sign. It is proposed that the measurement of tracheal microviscosity provides a method for checking pulmonary function in the first days of life. The method is rapid and sensitive; it detects small variations barely detectable using more conventional ways by which pulmonary phospholipids are usually studied.

Studies of ion transport across respiratory epithelia are of great interest if we are to understand the pathophysiology of diseases such as cystic fibrosis in which ion transport is abnormal. Concentrations of elements were determined in various subcellular regions of normal or isoproterenol-treated hamster tracheal epithelium, using X-ray microanalysis of freeze-dried cryosections. Samples of trachea were taken from animals under anesthesia and either frozen in situ or dissected and plunge frozen. Concentrations of Mg, P, S, Cl, K and Ca were higher in cytoplasm and nuclei of control epithelial cells in dissected samples than in cryoneedle samples. Following treatment with isoproterenol, a large decrease in the concentration of Cl was observed. The results confirm that cyclic AMP-regulated chloride secretion is unaffected by anesthesia.

The specificity of the High Iron Diamine-Alcian Blue pH 2.5 (HID-AB 2.5) procedure was examined in tissue sites containing sialogycoproteins alone or differing proportions of sialo- and sulphosialoglycoproteins. Studies with HID in differing final concentrations of hydrochloric acid or sodium chloride confirmed that staining is dependent upon both the pH and the ionic strength of the dye bath and demonstrated a marked heterogeneity in the pKa of the anionic groups of sialosulphoglycoproteins. Use of the sequence High Iron Diamine-Alcian Blue pH 1.0 demonstrated that complete or almost complete staining of O-sulphate esters occurred when HID was prepared in water (final pH 1.3). However, under these conditions HID-AB 2.5 was shown to be non-specific because only black HID staining was observed in sites containing large quantities of sialic acids. This non-specificity was due either to the masking of Alcian Blue staining by HID and/or the black HID staining of anionic groups other than sulphate. These results may account for some of the conflicting data obtained by different groups of investigators who have studied 'transitional mucosa' in human colonic diseases. Caution should be used in drawing conclusions from the use of HID-AB 2.5 without confirmatory evidence from other more specific procedures.

Application of cytochemical methods for complex carbohydrates at the light and electron microscopic levels served further to differentiate secretory cells of the rat tracheobronchial surface epitheliuim into serous and mucous categories, and permitted subclassification of serous cells into four types and of mucous cells into three types. The granules in different serous cells and Clara cells varied in staining with the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method, but generally lacked dialyzed iron (DI) affinity and apparently contained neutral glycoprotein. However, in some focal areas, serous and Clara cell granules showed a pattern of DI staining similar to the PA-TCH-SP reactivity. The PA-TCH-SP and DI methods stained granule glycoconjugate comparably in the three mucous cell types. The lack of high iron diamine (HID) affinity in these sites testified to carboxylated, periodate-reactive mucosubstance, presumably sialylated glycoprotein. The luminal surface of the apical plasmalemma of all surface epithelial cells stained with DI but lacked HID affinity. The PA-TCH-SP technique stained the surface of the apical plasmalemma of mucous cells, serous cells, and Clara cells in decreasing order of intensity. Microvilli of cililated cells were PA-TCH-SP-positive, but cilia were unreactive and apparently contained a glycosaminoglycan rather than the presumed sialylated glycoprotein of the other surfaces. DI and PA-TCH-SP reactivity of the apical far exceeded that of the basolateral plasmalemma in all surface epithelial cells. Two strata, presumed to be the lamina lucida and the lamina diffusa of the basement membrane under all the surface epithelial cells, stained with the DI but not the PA-TCH-SP method and apparently contained glycosaminogycan. Intraepithelial cells, interpreted as globule leukocytes, appeared to represent mast cells infiltrating the epithelium and carrying out endocytic activity.

Natural-abundance 13C NMR was used to study samples of native mucus from dog trachea and purified mucus glycoprotein from hog stomach. Despite the large molecular weight of the molecules (several million) and visco-elastic nature of the gel, spectra were produced which could be resolved into individual sharp resonances which gave significant structural detail. These results indicate the potential use of this powerful technique in the study of mucus glycoprotein structure in the undegraded molecule.

The fate of [3H]thymidine ([3H]Tdr) pulse-labelled cells was followed in tracheal epithelium of young male rats. The time course for cell differentiation, and the relation of events to tissue composition were studied. In vivo labelling and light microscope autoradiography of epoxy embedded sections were used. Labelled and total nuclei for each cell type, and combinations of labelled cells which were adjacent to one another, were tallied. Hierarchical analyses of variance were performed on the several data sets. All cell types, except ciliated, were labelled at 1 hr. A few labelled ciliated cells were seen 24 hr post-label. The frequency of labelled intermediate cells peaked at day 2; goblet and ciliated cells at day 3. No significant changes occurred in the labelling index, but at 24 hr the frequency of adjacent labelled cells (ALC) had increased greater than 5-fold, and changes had occurred in patterns of ALC combinations. The labelled ciliated cells which were seen at 24 hr were adjacent to labelled intermediate cells. No labelled basal-ciliated cell combinations were seen at any time. Data indicated that ciliated cells can develop from S-phase intermediate cells within 24 hr, and neither basal nor superficial goblet cells are progenitors of ciliated cells. It is proposed that both superficial goblet cell and ciliated cell development is preceded by two divisions: a basal cell division followed by an intermediate cell division.