Focalized proteolysis: spatial and temporal regulation of extracellular matrix degradation at the cell surface.

Cells respond to changes in their microenvironment by altering their cell surface and extracellular matrix proteins. Rapid and irreversible changes in these proteins are possible through their degradation or activation by proteolysis. By focalizing the proteolytic events at or near the cell surface, these processes can be effective even in the presence of high concentrations of inhibitors. Evidence is emerging that secreted and transmembrane matrix metalloproteinases, metalloproteinases of the adamalysin and astacin (tolloid) families, and serine proteinases are crucial in development, differentiation, cell motility and invasion, and cell-extracellular decisions.

Morphological studies of stimulated adrenergic axon varicosities in the mouse vas deferens.

The postganglionic axons of sympathetic neurons innervating the mouse vas deferens were stimulated transmurally in vitro by passing square pulses between two platinum electrodes. The ultrastructural appearance of the adrenergic nerve terminals was compared to samples fixed immediately after 30 min of stimulation and in samples allowed to recover for 2 h before fixation. The contralateral vasa deferentia served as controls, and these were incubated in Krebs solution for the same period as stimulated muscles. For each of four experiments, the mean number of large and small dense-core vesicles per square micrometer was calculated, as were the mean area and perimeter of the axon varicosities in each group. It was found that the number of small vesicles per square micrometer decreased by 60% during the stimulation period, but returned almost to control levels 2 h later. Large vesicles did not change in number during the stimulation or recovery periods. The proportion of vesicles containing cores was also determined for each group and found to decline just after stimulation in the small vesicle population, but to remain constant in the large vesicle population. The core depletion was partly reversed after 2 h. The vesicle recovery process was studied by use of the extracellular tracer horseradish peroxidase (HRP). When HRP was present in the extracellular space during stimulation, large numbers of vesicles contained the marker after recovery from stimulation. Thus, it is proposed that adrenergic axon varicosities recycle vesicle membrane through the plasma membrane in a manner similar to that already described for cholinergic nerve terminals.

Localization of Na pumps in the tracheal epithelium of the dog.

Binding of [3H]ouabain by the dog's tracheal epithelium shows a nonspecific component depending linearly on ouabain concentration, and a specific saturable component with a Km of 10(-7) M. Control experiments showed that the tracer taken up was not trapped within the extracellular space nor bound to tissue collagen. Inhibition of the saturable uptake by high K, metabolic inhibition, low Na, and low temperature indicated that binding was to Na/K ATPase. One-sided exposures of tissue sheets to tracer showed that the submucosal side took up 10 X as much tracer as the luminal. Autoradiography localized tracer uptake under all conditions to the cells' basolateral membranes.

Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells.

To investigate the hypothesis that neutrophil proteases stimulate airway gland secretion, we studied the effect of human cathepsin G and elastase on secretion of 35S-labeled macromolecules from cultured bovine airway gland serous cells. Both proteases stimulated secretion in a concentration-dependent fashion with a threshold of greater than or equal to 10(-10) M. Elastase was more potent than cathepsin G, causing a maximal secretory response of 1,810 +/- 60% over baseline at 10(-8) M. The maximal response to cathepsin G (1,810 +/- 70% over baseline at 10(-7) M) was similar to the maximal response to elastase. These responses were greater than 10-fold larger than the response to other agonists such as histamine. Protease-induced secretion was noncytotoxic and required catalytically active enzymes. The predominant sulfated macromolecule released by proteases was chondroitin sulfate proteoglycan. Immunocytochemical staining demonstrated chondroitin sulfate in cytoplasmic granules and decreased granular staining after stimulation of cells with elastase. The neutrophil proteases also degraded the proteoglycan released from serous cells. Cathepsin G and elastase in supernatant obtained by degranulation of human peripheral neutrophils also caused a secretory response. Thus, neutrophil proteases stimulate airway gland serous cell secretion of chondroitin sulfate proteoglycan and degrade the secreted product. These findings suggest a potential role for neutrophil proteases in the pathogenesis of increased and abnormal submucosal gland secretions in diseases associated with inflammation and neutrophil infiltration of the airways.

Human bronchus and intestine express the same mucin gene.

The amino acid and sugar composition of mucins from various organs is similar but not identical. This could arise by one or more of the following: organ-specific processing of a single core protein, organ-specific splicing of a single mucin mRNA, or organ-specific expression of various mucin genes. To begin to investigate the source of this variability, we examined (a) immunological cross-reactivity and (b) cDNA cross-hybridization, among several mucin-secreting organs of the human body. Peptide-directed antibodies raised against both nondeglycosylated (LS) and deglycosylated (HFB) intestinal mucin strongly stained mucous cells in the bronchial epithelium and submucosal glands, indicating homology between mucins of the bronchus and intestine at the peptide level. By screening a bronchus cDNA library with an intestinal mucin cDNA, SMUC-41, we isolated a bronchus mucin cDNA, HAM-1. This cDNA is 96% homologous to the first repeat of SMUC-41. HAM-1 hybridized to restriction fragments of human genomic DNA identical to those hybridizing to SMUC-41 on Southern blots. SMUC-41 also hybridized to polydisperse transcripts in the bronchus, cervix, gall bladder, and mammary gland, indicating mucin homology among all these organs at the RNA level. We conclude that the bronchus and intestine express a common mucin gene, which is likely co-expressed by at least several other mucin-secreting organs.

Characterization of quantitative mucin variants from a human colon cancer cell line.

Colonic mucins are high molecular weight glycoproteins produced by goblet cells of colonic epithelium. Some studies have indicated that patients with colonic cancers that produce high amounts of mucin have a poorer prognosis than patients whose tumors produce low amounts of mucin. At present, however, the role of mucin in affecting the behavior of colon cancer cells is not well understood. To further elucidate the relationship between cellular mucin content and the growth characteristics and morphology of tumor cells, we utilized a replica plating technique and immunoscreening method to identify and purify variant clones of the human colon cancer cell line LS174T that produce high and low levels of mucin. This procedure enabled us to isolate two high mucin-containing variants (HM3 and HM7) and one low mucin-containing variant (LM12). These variants exhibited different morphology. Both high mucin variants tended to form cell aggregates and suspended cells with adjoining mucoid threads. The low mucin variant formed spread monolayers on the substratum with the formation of cell processes. Metabolic labeling using [3H]glucosamine demonstrated that high mucin variants synthesized 2-fold more mucin in the cell layer and secreted 3-fold more mucin into the culture medium than the low mucin variant. The colony-forming efficiency in semisolid agar for these variants positively correlated with their mucin content. High mucin variant cells when injected into athymic nude mice formed tumors 2-fold larger than those of the parental cells while the low mucin variant formed tumors only one-half as large as those of the parental cell line. These mucin variants should provide a useful model for understanding the biological behavior of mucinous colon cancer cells in vivo and in vitro.

The architecture of nerves and ganglia of the ferret trachea as revealed by acetylcholinesterase histochemistry.

The goal of this study was to determine the architecture of the nerves and ganglia of the ferret trachea. Tracheas from four newborn ferrets and three adult ferrets were stained histochemically for acetylcholinesterase activity and analyzed in their entirety as whole mounts. The architecture consisted of one or two longitudinal nerve trunks overlying the posterior surface of the trachealis muscle, a dense plexus of nerves superficial to the trachealis muscle that interconnected these longitudinal nerve trunks, and, on the anterior surface, a plexus superficial to the submucosal glands and located between the cartilaginous rings. In addition, deep neural plexuses were associated with the trachealis muscle and with the submucosal glands. Ganglion cell bodies along the longitudinal nerve trunks were large (mean diameter +/- S.E. = 34.3 +/- 0.3 microns), were usually attached to the nerve trunk by a stalk, and were loosely clustered in groups of as many 38 cell bodies. By contrast, those cell bodies of the superficial muscle and gland plexuses were significantly smaller (mean diameter +/- S.E. = 24.2 +/- 0.3 microns), were never attached by a stalk, and were tightly clustered in ganglia of one to four cell bodies. We conclude that nerves and ganglia of the ferret trachea constitute one or two longitudinal nerve trunks containing ganglia with large cell bodies, two superficial nerve plexuses containing ganglia with small cell bodies overlying the smooth muscle and submucosal glands, respectively, and two deep nerve plexuses providing the terminal innervation to the muscle and glands.

Altered mucin expression is a field change that accompanies mucinous (colloid) breast carcinoma histogenesis.

Mucinous carcinomas of the breast, so-called colloid carcinomas, exhibit better prognoses than their nonmucinous breast counterparts. This biological difference exhibited by mucinous breast carcinomas prompted us to examine the relationship of mucin expression to colloid carcinoma histogenesis. We studied 50 colloid carcinomas, 50 noncolloid cancers, and 50 normal breasts by hematoxylin-eosin (H&E) and Alcian blue staining, mucin immunohistochemistry, in situ hybridization with a battery of MUC riboprobes, and ancillary digital image analysis. We observed luminal mucin in normal ducts in 80% of colloid carcinomas compared with 10% of noncolloid carcinomas and 6% of normal breasts (P < .01). In the cases of colloid carcinoma that showed mucin-filled ducts, luminal mucin was observed in 40% of the normal ducts and acini, 40% to 75% of the ducts involved by hyperplasia, atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS), respectively, and in 50% of the co-incidental areas of cysts (mucoceles), adenosis, fibroadenoma, and intraductal papilloma (P < .01). Immunohistochemistry showed that colloid carcinomas showed strong MUC2 cytoplasmic immunoreactivity and decreased MUC1 immunoreactivity compared with noncolloid carcinomas. In situ hybridization studies indicated fivefold increased MUC2 signals and twofold increased MUC5 signals within adjacent and remote normal epithelium in only the colloid carcinoma cases (P < .01; P < .05). In these cases of colloid carcinoma, these increased MUC2 and MUC5 signals were also observed in areas of hyperplasia, ADH, DCIS, and invasive carcinoma. In contrast, the noncolloid carcinomas showed fivefold increased MUC1 signals but no increases in MUC2 or MUC5. In mixed colloid/noncolloid carcinomas, the colloid areas had identical mucin expression patterns as the pure colloid carcinomas, but there was a loss of MUC2 and MUC5 expression and a gain of MUC1 expression in the noncolloid areas that was therefore identical to the pattern observed in pure noncolloid carcinoma. In this study, we conclude that the altered expression of mucin so characteristic of colloid carcinoma is also a field change present in adjacent and remote normal breast epithelium.

In vivo activity of tracheal parasympathetic ganglion cells innervating tracheal smooth muscle.

In vivo intracellular recording and intrasomal injection of Lucifer yellow revealed two populations of postganglionic parasympathetic neurons in the tracheal ganglia of cats. One consisted of large cells that had an inspiratory rhythm, had a significant post-spike afterhyperpolarization, and projected to the tracheal smooth muscle. The second consisted of small cells that fired with an expiratory rhythm, had no significant afterhyperpolarization, and projected to the intercartilaginous spaces.

Muscarinic receptors in lung and trachea: autoradiographic localization using [3H]quinuclidinyl benzilate.

[3H]Quinuclidinyl benzilate binding to slide-mounted frozen sections of ferret lung was of high affinity (KD 63 +/- 14 pM, mean +/- S.E., n = 4), and characteristic of interaction with muscarinic cholinergic receptors. Light microscopic autoradiography showed muscarinic receptors to be localized predominantly to smooth muscle of trachea and intrapulmonary cartilaginous airways, and to submucosal glands. There was much less labelling of bronchiolar smooth muscle, airway epithelium and vascular smooth muscle and no labelling of alveoli. This distribution of receptors parallels that of cholinergic innervation.

Pulmonary alpha-adrenoceptors: autoradiographic localization using [3H]prazosin.

We determined the distribution of pulmonary alpha-adrenoceptors by autoradiographic localisation of [3H]prazosin binding to frozen sections of ferret lung. Specific binding of [3H]prazosin to lung sections was saturable and of high affinity (KD = 0.44 +/- 0.55 nM; mean +/- S.E., n = 5), with a specificity indicating binding to alpha 1-receptors. Autoradiographic showed that alpha 1-receptors were present in highest density in vascular smooth muscle (small vessels greater than large vessels), and were also present in airway submucosal glands and epithelium. There was also scanty labelling of alveolar walls which may be to contractile interstitial cells (Kapanci cells). Although smooth muscle of bronchi showed little labelling, surprisingly that of bronchioles was heavily labelled. The high density of alpha-receptors in small airways may be relevant to asthma in which alpha-adrenergic responses are activated. This method offers a means by which autonomic receptors of small airways may be investigated without the confusing contribution of other contractile elements and larger airways.

Transmission in airway ganglia of ferrets: inhibition by norepinephrine.

We examined the possibility that norepinephrine inhibits transmission in parasympathetic ganglia of the ferret trachea. We impaled ganglion cells on recording microelectrodes and evoked postsynaptic action potentials by stimulating fiber tracts entering the ganglion. When norepinephrine was added to the recording bath, the action potentials were blocked. Phentolamine reversed this block. These results indicate that, by activating alpha-receptors, norepinephrine inhibits transmission in airway ganglia.

Regulation of airway secretory cells.

Inhaled particles are cleared from the airways by ciliary transport of a discontinuous mucous layer. The effectiveness of this process depends on the viscoelastic properties of the secretion, which in turn depend on the composition and interaction of the glycoprotein constituents. In human airways, two major cell types in the surface epithelium (goblet and ciliated) and two in the submucosal glands (serous and mucous) are known to contain mucin- and/or serum-type glycoproteins. This article summarizes current knowledge regarding the secretable products of each of these cell types and the conditions under which they are released into the airway lumen.

Tracheal gland mucous cells stimulated in vitro with adrenergic and cholinergic drugs.

To determine the responsiveness of tracheal mucous cells to adrenergic and cholinergic stimulation, we analyzed changes in their structure induced by neurotransmitter-like agonists. Ferret tracheal rings were exposed for 30 min in vitro to one of the following: phenylephrine, isoproterenol, or bethanechol (all at 10(-5) M), in the presence of absence of appropriate antagonists. Electron microscopy and morphometric analysis revealed that the volume density of mucous cells (Vvmc, i.e. the space occupied by mucous cells in the submucosa) significantly decreased, and the surface density of mucous cell apical membrane (Svam) increased in response to isoproterenol and bethanechol but not to phenylephrine. In metabolic labeling experiments, the morphological changes were accompanied by secretagogue-evoked release of 35S-labeled macromolecules. Taken together, these data suggest that tracheal mucous cells secrete 35S-labeled macromolecules in response to beta-adrenergic and muscarinic agonists by an exocytotic process that involves a reduction in cell size.

Studies of tracheal secretion using serous cell cultures and monoclonal antibodies.

The glycoconjugate composition of tracheal secretions varies with physiological and pathophysiological parameters. Believing that these differences might be explained by metabolic or regulatory modifications of particular cell types, we have developed strategies for biochemical analysis at the cellular level. We have produced monoclonal antibodies whose determinants are restricted to a single secretory cell type (serous, mucous, or goblet cell granules, or ciliated cell glycocalyx). By enzyme immunoassay (ELISA), we have characterized four of the antibodies biochemically, and have also used the antibodies as quantitative molecular probes to detect release of antigen from mixed cell explants. Four of the antigens are carried by carbohydrate moieties of high molecular weight glycoproteins. Western blot analysis shows their molecular weight in reducing gels (SDS-PAGE) to exceed 200 kD. When used in parallel with pulse-chase labeling studies, the antibodies are both more sensitive and specific (than bound radioactivity) in detecting gland or goblet cell secretion in response to autonomic drugs or proteases. We have also isolated and cultured serous gland cells for physiological and biochemical studies. These cells express serous cell phenotype as reflected by ultrastructure, histochemistry, and lysozyme activity. Biochemical analysis of their secretory products reveals glycoconjugate components which are heterogeneous with respect to both molecular weight and charge. Radiolabeled secretory products eluting in the void volume of Sepharose C1 4B were completely degraded by chondroitinase ABC. This indicates that the major glycoconjugate produced by serous cell is a proteoglycan resembling chondroitin sulfate.

Regulation of secretion from serous and mucous cells in the trachea.

The physical properties of mucus and the efficiency of tracheal mucociliary clearance depend on maintenance of a balanced interaction among several epithelial cell types. Some of these cell types are specialized to perform ion and water transport, others to perform synthesis and secretion of macromolecules. Our studies have been aimed specifically at identifying the neural mechanisms regulating macromolecule secretion from two of these cell types, i.e. serous and mucous gland cells. Because these cells occur as part of a complex epithelium, it is difficult to monitor the properties and functions of each cell type individually. We have therefore relied principally on morphological methods, which can potentially focus on a single cell type within a heterogeneous tissue. Such studies, however, depend on the availability of visible markers (enzyme-labelled antibodies, radioligands, etc.), and many important aspects of gland cell function cannot be assessed morphologically. Two alternative approaches are therefore being developed: the isolation and segregation of gland cells according to type, and the production of monoclonal antibodies that recognize secretory products of individual cell types. These methods allow serous and mucous cells to be studied by biochemical as well as morphological methods.

Monoclonal antibodies directed against human airway secretions. Localization and characterization of antigens.

Cellular mechanisms of normal airway mucus secretion and their alterations in chronic obstructive lung disease are poorly understood. To aid in their study, the authors have produced a panel of monoclonal antibodies directed against various constituents of human airway secretions. Two fusions yielded 401 hybridoma-containing cultures. Supernatants from 150 of these cultures stained human tracheal secretory cells by immunofluorescence. Twenty-nine hybridomas were selected for expansion because they selectively stained a single cell type or displayed another interesting distribution. Antigens were further characterized by their localization in glycol methacrylate sections of human trachea, sensitivity to periodate oxidations, selective affinity for fraction peaks obtained by Sepharose 4B chromatography, and reactivity with molecules of various sizes, as estimated by SDS-PAGE. These antibodies will be useful for 1) quantitative detection of antigens in sputum or lavage samples by immunoassay and 2) purification and biochemical characterization of molecular constituents of airway secretions in health and disease.

Airway secretion: a cell-specific analysis.

Respiratory mucus is a complex secretion elaborated by several cell types in the airway wall: serous and mucous gland cells in the submucosa, and ciliated and goblet cells in the epithelium. The cell types are under independent regulatory mechanisms, most of which are poorly understood. Two new approaches are described which permit analysis of these mechanisms and the role of each cell type in mucus production. Using cell-specific monoclonal antibodies, we have obtained biochemical information about mucus components contributed by the various cell types. Antibodies are also being used in enzyme immunoassays (ELISA) to detect cell-specific secretion from mixed cell biopsies. In other studies, analysis of secretory products from pure cultures of serous gland cells has revealed that the major glycoconjugates released by this cell type are chondroitin sulfate proteoglycans, hyaluronic acid and N-linked glycoproteins of complex type.

Mucin in disease. Modification of mucin gene expression in airway disease.

Mucus hypersecretion is a characteristic feature of several human airway diseases, including chronic bronchitis, cystic fibrosis, and asthma. Although its pathogenesis is poorly understood, hypersecretion apparently results from the abnormally large number of mucous cells found in hypersecretory airways. The factors giving rise to these mucous cells are unknown, but experimental evidence supports possible roles for both mitosis (mucous cell hyperplasia) and differentiation (mucous cell metaplasia). On the basis of the hypothesis that differentiation would require activation of mucin mRNA transcription, we have used mucin cDNA to monitor mucin mRNA levels in an animal model of chronic bronchitis. We first showed that a mucin gene (SMUC or MUC-2) cloned from the human intestine is also expressed in the human airways and is the same or homologous to genes expressed in other human mucin-producing organs. We next showed that a homologue of the SMUC gene is expressed in several animal species, including the rat. Finally, we showed that the induction of experimental chronic bronchitis by SO2 in rats is accompanied by the induction (from near zero baseline) of airway mucin mRNA. The induction by irritants of high steady-state levels of mucin mRNA may represent one of the early events in mucous cell differentiation and hypersecretion.

Mapping of adrenergic receptors in the trachea by autoradiography.

We investigated the distribution of adrenergic receptors in ferret trachea using autoradiography. [3H]Dihydroalprenolol, used to identify beta-adrenoceptors, revealed a high density of specific binding sites over surface epithelium and submucosal glands, with less labelling of smooth muscle. [3H]prazosin labelling showed that alpha 1-receptors were numerous in glands and epithelium, but sparse in smooth muscle. Comparison of adrenergic receptor densities in tracheal sections from the same animals showed a rank order for submucosal glands of alpha 1 greater than beta, for epithelium beta greater than alpha 1 and for smooth muscle beta greater than alpha 1. Within the submucosal glands, alpha 1- and beta-adrenergic receptors were differentially distributed, with alpha 1-receptors being significantly more numerous over serous than mucous cells and beta receptors being significantly more numerous over mucous than serous cells. This technique provides insight into adrenergic regulation of airway function and should be useful in investigations of how relative receptor densities may be altered in disease.