Biomedical subjects

C B Basbaum

Publications and source records attributed to C B Basbaum.

At least 19 recordsLinked to original sources

PubMed1992

Bovine tracheal serous cell secretion: role of cAMP and cAMP-dependent protein kinase.

The role of adenosine 3',5'-cyclic monophosphate (cAMP) and protein phosphorylation during beta-adrenergic receptor stimulation of bovine tracheal gland serous cells was investigated in vitro. Isoproterenol, a beta-adrenergic agonist, increased the secretion of 35S-labeled molecules. Intracellular cAMP levels were increased within 1 min after stimulation of bovine tracheal gland serous cells with isoproterenol. The dose-response relationship for isoproterenol-stimulated generation of cAMP correlated with the dose-response relationship for isoproterenol-stimulated secretion of 35S-labeled molecules. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine potentiated both isoproterenol-evoked secretion of 35S-labeled molecules and the production of intracellular cAMP, and the beta-adrenergic receptor antagonist propranolol completely blocked both effects. The secretory response of the cells to isoproterenol could be mimicked by the cAMP analogues 8-bromoadenosine 3',5'-cyclic monophosphate and dibutyryl adenosine 3',5'-cyclic monophosphate. Activity of cAMP-dependent kinase was measured in soluble and particulate cell extracts. cAMP effected the state of phosphorylation of proteins associated with the soluble but not the particulate fraction. These studies are consistent with the hypothesis that beta-adrenergic stimulation of secretion from bovine tracheal gland serous cells occurs via a cAMP-mediated pathway and that one of the molecular events in this pathway is cAMP-dependent protein phosphorylation.

1-Methyl-3-isobutylxanthine↗

PubMed1992

Extracellular matrix proteins regulate morphologic and biochemical properties of tracheal gland serous cells through integrins.

The extracellular matrix has been shown to influence the differentiation of epithelial cells. To identify cues from the extracellular matrix controlling the differentiation of tracheal gland serous cells, we examined the effects of culturing these cells on various extracellular matrix proteins. Bovine tracheal gland (BTG) serous cells attached to Type IV collagen (COL IV), laminin (LM), and fibronectin (FN) in a concentration-dependent manner. Morphologic analysis showed that cells formed confluent monolayers on COL IV or LM, whereas on FN, cells formed birefringent spheres. Metabolic labeling experiments showed that [35S]methionine-labeled protein bands at 68, 105, and 120 kD were prominent when cells were grown on COL IV or LM, but were lost or reduced when the cells were grown on FN. COL IV also enhanced the expression of proteins at 14, 16.5, 18, and 21.5 kD. Attachment to all substrates was inhibited by an antibody directed against beta 1 integrins. This antibody precipitated several integrin heterodimers from a BTG cell membrane extract, caused partial retraction of cells from all substrates, and strongly suppressed the expression of COL IV- and LM-dependent proteins. Control experiments indicated that the latter did not require conspicuous changes in cell shape. These results show that some biochemical properties of serous cells are regulated by integrin-mediated effects of extracellular matrix proteins in vitro and suggest that similar regulation may occur during normal development and remodeling of the glands in vivo.

Albumins↗

PubMed1991

Regulation of secretion in cultured tracheal serous cells by protein kinases A and C.

We recently reported that cultured gland serous cells release chondroitin sulfate proteoglycans (CSPGs) in response to beta-adrenergic agonists. In this study, we analyzed this regulatory pathway and other cellular mechanisms responsible for CSPG secretion. We show the following. 1) Isoproterenol increased CSPG secretion in a concentration-dependent manner, with maximal stimulation (50%) obtained at 10(-5) M; at this concentration, the beta-agonist also stimulated protein kinase A (PKA) by 50%, whereas it increased cellular adenosine 3',5'-cyclic monophosphate (cAMP) content by 300%. 2) Phenylephrine (10(-5) M), 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (1.6 x 10(-7) M), and A23187 (10(-6) M) also stimulated CSPG secretion; this stimulation was concomitant with protein kinase C (PKC) translocation from cytosol to membrane, was blocked by sphingosine (2 x 10(-5) M), and was additive with that elicited by isoproterenol. 3) All PKC activators potentiated the isoproterenol-induced increased in cAMP accumulation without modifying the activation of PKA elicited by the beta-agonist. Our results indicate that although the signaling pathways triggered by alpha- and beta-adrenergic agonists converge at the level of adenylate cyclase in tracheal serous cells, PKA and PKC independently regulate CSPG secretion.

Animals↗

PubMed1991

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.

Animals↗

PubMed1991

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.

Amino Acid Sequence↗

PubMed1990

Long-term culture of normal and cystic fibrosis epithelial cells grown under serum-free conditions.

The understanding of pathways associated with differentiated function in human epithelial cells has been enhanced by the development of methods for the short-term culture of human epithelial cells. In general these methods involve the use of serum. The subculture and maintenance of epithelial cells in long-term culture has been more problematic. A serum-free medium developed for human bronchial epithelial cells was slightly modified and found to be useful for the subculture and long-term maintenance of not only bronchial epithelial cells, but also tracheal, nasal polyp, and sweat gland epithelial cells from either normal or cystic fibrosis individuals. The cells maintained epithelial-specific characteristics after multiple subcultures. Monolayers of epithelial cells showed junctional complex formation, the presence of keratin, and micro villi. Functional studies with Ussing chambers showed short circuit current (Isc) responses to isoproterenol, bradykinin, or calcium ionophore (A23187) in subcultured tracheal and bronchial cells.

Bronchi↗

PubMed1990

The serous cell.

Explore the source record for details and available documents.

Animals↗

PubMed1990

Receptors on airway gland cells.

Airway submucosal glands are by volume the most important source of macromolecules in airway secretions. These secretions, containing gel-forming mucins, antibacterial proteins, and antiproteases, comprise the major defensive barrier protecting the host against airborne pathogens. The identification of the mechanisms regulating secretion from the submucosal glands is key to understanding the genesis of this barrier and how it is altered by disease processes. Using a variety of methods, we and others have identified on the gland cells of several species receptors specific for ACh, norepinephrine, substance P, VIP, PGE1, PGE2, PGA1, PGD2, histamine and bradykinin. These receptors all participate in modulating the secretory activity of the airway submucosal glands. Studies of homogeneous cultures of bovine airway serous cells have yielded detailed information regarding the beta-adrenergic receptor on these cells. Using radioligand binding techniques, we found evidence for the presence of a single high affinity beta receptor of beta-2 subtype. Occupancy of this receptor by isoproterenol causes an elevation in the concentration of intracellular cAMP, which in turn stimulates the phosphorylation of a subset of cytoplasmic and membrane proteins. Based on the kinetics and pharmacology of these effects, it is likely that cAMP functions as a second messenger in the serous cell secretory pathway, probably acting through protein kinases. Current efforts are directed at identification of those phosphoproteins whose phosphorylation and dephosphorylation times are consistent with their possible roles in secretion.

Animals↗

PubMed1990

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.

Animals↗

PubMed1989

Mast cell chymase. A potent secretagogue for airway gland serous cells.

Submucosal glands are the major sources of airway secretions in most mammals. Mast cells are abundant in the environment of airway submucosal glands and are rich sources of secreted proteases. To investigate the hypothesis that mast cell proteases stimulate airway gland secretion, we studied the ability of the two major mast cell granule proteases, chymase and tryptase, to cause secretion of 35S-labeled macromolecules from a line of cultured bovine airway gland serous cells. Mast cell chymase and tryptase were purified from dog mastocytoma cells. Chymase markedly stimulated serous cell secretion in a concentration-dependent fashion with a threshold of 10(-10) M, whereas tryptase had no effect. The response to 10(-8) M chymase (1530 +/- 80% over base line) was approximately 10-fold higher than that evoked by other agonists such as histamine and isoproterenol. The predominant 35S-labeled macromolecule released by chymase was chondroitin sulfate proteoglycan, the glycoconjugate present in serous cell secretory granules. The response to chymase was non-cytotoxic and was blocked by active site inhibitors of chymase (soybean trypsin inhibitor and chymostatin) and by inhibitors of cellular energy metabolism (azide,2,4-dinitrophenol, dicumarol). Supernatant obtained by degranulation of mastocytoma cells caused a secretory response of comparable magnitude to that caused by chymase. These findings demonstrate that chymase, but not tryptase, is a potent secretagogue for airway gland serous cells, and they suggest a possible role for chymase-containing mast cells in the pathogenesis of airway hypersecretion.

Animals↗

PubMed1989

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.

Animals↗

PubMed1989

Characterization of beta-adrenergic receptors in cultured bovine tracheal gland cells.

We characterized the beta-adrenergic receptors that mediate secretory responses to isoproterenol in cultured bovine tracheal submucosal gland cells. Previous studies have shown that these cells have morphological and biochemical features characteristic of serous cells. Isoproterenol, epinephrine, and norepinephrine each stimulated the secretion of 35SO4-labeled macromolecules from these cultured serous cells with a rank order of potency (isoproterenol greater than epinephrine greater than norepinephrine) consistent with the presence of beta 2-adrenergic receptors. These functional studies were supported by radioligand-binding studies using [I125]-iodocyanopindolol (125I-CYP) to identify beta-adrenergic receptors. 125I-CYP binding to membrane particulates prepared from cultured serous cells was saturable and of high affinity (equilibrium dissociation constant 20 +/- 3 pM; mean +/- SE, n = 6) and was antagonized stereoselectively by propranolol. Adrenergic agonists competed for 125I-CYP-binding sites with a rank order of potency characteristic of the beta 2-adrenergic receptor subtype. A specific beta 2-adrenergic receptor antagonist, ICI 118.551, competed for a single class of 125I-CYP-binding sites with high affinity (inhibition constant 1.8 +/- 0.3 nM, n = 3). We concluded that the secretory response of cultured tracheal gland cells to isoproterenol is a response mediated by beta-adrenergic receptors of the beta 2 subtype.

Animals↗

PubMed1988

Synthesis and secretion of an albumin-like protein by cultured bovine tracheal gland serous cells.

Bovine tracheal submucosal gland serous cells were incubated with (35S) methionine. The proteins synthesized and secreted into the culture medium during pulse and chase periods were analyzed. Three major protein bands with apparent molecular weight values of 67 kD, 47 kD and 32 kD were detected by fluorography following SDS-polyacrylamide gel electrophoresis. Based on the molecular weight, immuno-reactive cross reactivity and surface molecular change characteristics, the labeled Mr 67 kD protein was identified as an albumin-like protein. Our results suggest that albumin found in airway secretions can originate, at least in part, from tracheal gland serous cells.

Albumins↗

PubMed1988

Glycoconjugates secreted by bovine tracheal serous cells in culture.

Glycoconjugates secreted by bovine tracheal gland serous cells in culture were characterized after incorporation of radioactive precursor [1-14C]glucosamine and stimulation with isoproterenol. Under dissociative conditions, glycoconjugates eluted in both the void and included volumes on Sepharose Cl-4B. Fractionated by anion-exchange chromatography, the high-molecular-weight (Sepharose Cl-4B; V0) glycoconjugates gave two acidic fractions eluting at 0.5 and 2.0 M NaCl; low-molecular-weight glycoconjugates of the included volumes gave a neutral fraction and two acidic fractions eluting at 0.5 and 2.0 M NaCl. Based on chemical analysis and specific enzymatic digestions, the material eluting in the void volume was shown to contain hyaluronic acid and chondroitin sulfate proteoglycan. In addition, the presence of small amounts of galactose, fucose, sialic acid, glucosamine, and galactosamine suggest the presence of O-glycosidically linked glycoproteins in the void volume. The identification of galactosaminitol in beta-eliminated oligosaccharides from this material confirms this notion. The material eluting in the included volume was shown to contain N-linked glycoproteins with glycans of complex type in the neutral fraction and chondroitin sulfate proteoglycans in the two acidic fractions. Significant N-sulfation of amino sugars was detected in the 0.5 M acidic fraction, indicating the presence of heparan sulfate. Hyaluronic acid and chondroitin sulfate proteoglycan have recently been identified in tracheal secretions; our results suggest that these components originate at least in part from tracheal gland serous cells.

Animals↗

PubMed1988

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.

Animals↗

PubMed1988

Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40.

To facilitate understanding of the mechanisms underlying pulmonary diseases, including lung cancer and cystic fibrosis, we have transformed and characterized cultures of human tracheal epithelial cells. Cells were transfected by calcium phosphate precipitation with a plasmid containing a replication-defective simian virus 40 (SV40) genome. Colonies of cells with enhanced growth potential were isolated and analyzed for transformation- and epithelial-specific characteristics. Precrisis cells were observed to express the SV40 large tumor antigen, produce cytokeratins, have microvilli, and form tight junctions. After crisis, cells continued to express the SV40 large tumor antigen as well as epithelial-specific cytokeratins and to display the apical membrane microvilli. Apical membrane Cl channels were opened in postcrisis cells exposed to 50 microM forskolin. These channels showed electrical properties similar to those observed in primary cultures. The postcrisis cells have been in culture for greater than 250 generations and are potentially "immortal." In addition to providing a useful in vitro model for the study of ion transport by human airway epithelial cells, the cells can be used to examine stages of neoplastic progression.

Cell Line, Transformed↗

PubMed1988

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.

Antibodies, Monoclonal↗

PubMed1987

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.

Animals↗