Establishment and characterization of a telomerase immortalized human gingival epithelial cell line.

BACKGROUND AND OBJECTIVE: Gingival keratinocytes are used in model systems to investigate the interaction between periodontal bacteria and the epithelium in the initial stages of the periodontal disease process. Primary gingival epithelial cells (GECs) have a finite lifespan in culture before they enter senescence and cease to replicate, while epithelial cells immortalized with viral proteins can exhibit chromosomal rearrangements. The aim of this study was to generate a telomerase immortalized human gingival epithelial cell line and compare its in vitro behaviour to that of human GECs. MATERIAL AND METHODS: Human primary GECs were immortalized with a bmi1/hTERT combination to prevent cell cycle triggers of senescence and telomere shortening. The resultant cell-line, telomerase immortalized gingival keratinocytes (TIGKs), were compared to GECs for cell morphology, karyotype, growth and cytokeratin expression, and further characterized for replicative lifespan, expression of toll-like receptors and invasion by P. gingivalis. RESULTS: TIGKs showed morphologies, karyotype, proliferation rates and expression of characteristic cytokeratin proteins comparable to GECs. TIGKs underwent 36 passages without signs of senescence and expressed transcripts for toll-like receptors 1-6, 8 and 9. A subpopulation of cells underwent stratification after extended time in culture. The cytokeratin profiles of TIGK monolayers were consistent with basal cells. When allowed to stratify, cytokeratin profiles of TIGKs were consistent with suprabasal cells of the junctional epithelium. Further, TIGKs were comparable to GECs in previously reported levels and kinetics of invasion by wild-type P. gingivalis and an invasion defective ΔserB mutant. CONCLUSION: Results confirm bmi1/hTERT immortalization of primary GECs generated a robust cell line with similar characteristics to the parental cell type. TIGKs represent a valuable model system for the study of oral bacteria interactions with host gingival cells.

Role of calcium and PKC in salivary mucous cell exocrine secretion.

Fluid and exocrine secretion of mucins by salivary mucous glands is regulated predominantly by parasympathetic activation of muscarinic receptors. A direct role for subsequent putative signaling steps, phospholipase C (PLC), increased intracellular calcium ([Ca(2+)](i)), and isoforms of protein kinase C (PKC) in mediating muscarinic exocrine secretion has not been elucidated, and these are potential therapeutic targets to enhance mucin secretion in hyposalivary patients. We found that muscarinic-induced mucin secretion by rat sublingual tubulo-acini was dependent upon PLC activation and the subsequent increase in [Ca(2+)](i), and further identified a transient PKC-independent component of secretion dependent upon Ca(2+) release from intracellular stores, whereas sustained secretion required entry of extracellular Ca(2+). Interactions among carbachol, PKC inhibitors, phorbol 12-myristate 13-acetate, and thapsigargin to modulate [Ca(2+)](i) implicated conventional PKC isoforms in mediating sustained secretion. With increasing times during carbachol perfusion of glands, in situ, PKC-α redistributed across glandular membrane compartments and underwent a rapid and persistent accumulation near the luminal borders of mucous cells. PKC-ß1 displayed transient localization near luminal borders, whereas the novel PKCs, PKC-δ or PKC-ε, displayed little or no redistribution in mucous cells. Collective results implicate synergistic interactions between diacylglycerol (DAG) and increasing [Ca(2+)](i) levels to activate cPKCs in mediating sustained muscarinic-induced secretion.

A mouse caries model and evaluation of aqp5-/- knockout mice.

Current techniques to alter gene expression in mice allow direct analysis of the net role of a host factor in caries development. Towards this goal we first established protocols to induce and score caries in NFS/N mice and determined caries susceptibility in mice with targeted deletion of the gene encoding aquaporin-5 (Aqp5-/-), a water channel involved in the production of saliva. In the NFS/N strain of mice total sulcal caries and severity scores were consistent between experiments, whereas smooth surface caries scores were lower, more variable but distributed fairly evenly among the buccal, lingual and sulcal surfaces. In Black Swiss/129SvJ mice (genetic background of Aqp5-/- mice) caries scores were 50-75% lower compared to NFS/N mice, suggesting strain variation in caries susceptibility under our experimental conditions. In Aqp5-/- mice, in which the volume of total salivary secretion is reduced by 60-65%, there was a significant increase in caries, primarily on the buccal and sulcal surfaces. Results indicate that caries susceptibility increases with a reduced salivary flow that is associated with decreased water content of saliva.

The gene encoding mouse Muc19: cDNA, genomic organization and relationship to Smgc.

We previously demonstrated expression of full-length transcripts for sublingual mucin apoprotein, Muc19, of approximately 24 kb (Fallon MA, Latchney LR, Hand AR, Johar A, Denny PA, Georgel PT, Denny PC, and Culp DJ. Physiol Genomics 14: 95-106, 2003). We now describe the complete sequence and genomic organization of the apomucin encoded by 43 exons. Southern analyses indicate a central exon of approximately 18 kb containing 36 tandem repeats, each encoding 163 residues rich in serine and threonine. Full-length transcripts are an estimated 22,795 bp in length that span 106 kb of genomic DNA. The transcriptional start site is 24 bp downstream of a TATA box and 42 bp upstream of the conceptual translational start codon. The putative apoprotein has an estimated mass of 693.4 kDa and contains 7,524 amino acids (80% serine, threonine, glycine, alanine, and proline). We present a model for rat Muc19 transcripts and compare the conceptually translated Muc19 proteins for mouse, rat, pig, and the 3' end of human Muc19. Conserved among these apoproteins are a signal peptide, a large tandem repeat region, von Willebrand factor type C and D domains, a trypsin inhibitor-like Cys-rich domain, and a COOH-terminal cystine knot-like domain. Southern blot analyses indicate transcripts for Muc19 and Smgc (submandibular gland protein C) are splice variants of a larger gene, Muc19/Smgc. Comparative Northern analyses between the major salivary glands demonstrate highly selective Muc19 expression in neonatal and adult sublingual glands, whereas Smgc is expressed in neonatal submandibular and sublingual glands. Regulation of Muc19/Smgc gene expression is discussed with respect to alternative splicing and mucous cell cytodifferentiation.

The sld mutation is specific for sublingual salivary mucous cells and disrupts apomucin gene expression.

NFS/N-sld mice harbor a spontaneous autosomal recessive mutation, sld (sublingual gland differentiation arrest) and histologically display attenuated mucous cell expression in sublingual glands (Hayashi et al. Am J Pathol 132: 187-191, 1988). Because altered serous demilune cell expression is unknown, we determined the phenotypic expression of this cell type in mutants. Moreover, we evaluated whether absence of glycoconjugate staining in 3-day-old mutant glands is related to disruption in apomucin gene expression and/or to posttranslational glycosylation events. Serous cell differentiation is unaffected, determined morphologically and by serous cell marker expression (PSP, parotid secretory protein; and Dcpp, demilune cell and parotid protein). Conversely, apical granules in "atypical" exocrine cells of mutant glands are PSP and mucin negative, but contain abundant SMGD (mucous granule marker). Age-related appearance of mucous cells is associated with expression of apomucin gene products, whereas SMGD expression is unaltered. "Atypical" cells thus appear specified to a mucous cell fate but do not synthesize mucin glycoproteins unless selectively induced postnatally, indicating the sld mutation disrupts apomucin transcriptional regulation and/or decreases apomucin mRNA stability.

G protein coupling to M1 and M3 muscarinic receptors in sublingual glands.

Rat sublingual gland M2 and M3 muscarinic receptors each directly activate exocrine secretion. To investigate the functional role of coreceptor expression, we determined receptor-G protein coupling. Although membrane proteins of 40 and 41 kDa are ADP-ribosylated by pertussis toxin (PTX), and 44 kDa proteins by cholera toxin (CTX), both carbachol-stimulated high-affinity GTPase activity and the GTP-induced shift in agonist binding are insensitive to CTX or PTX. Carbachol enhances photoaffinity labeling ([alpha-(32)P]GTP-azidoaniline) of only 42-kDa proteins that are subsequently tractable to immunoprecipitation by antibodies specific for Galpha(q) or Galpha(11) but not Galpha(12) or Galpha(13). Carbachol-stimulated photoaffinity labeling as well as phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis is reduced 55% and 60%, respectively, by M1 receptor blockade with m1-toxin. Galpha(q/11)-specific antibody blocks carbachol-stimulated PIP2 hydrolysis. We also provide estimates of the molar ratios of receptors to Galpha(q) and Galpha(11). Although simultaneous activation of M1 and M3 receptors is required for a maximal response, both receptor subtypes are coupled to Galpha(q) and Galpha(11) to stimulate exocrine secretion via redundant mechanisms.

In vitro studies of the regulation of mucous gland secretion.

Our laboratory is concerned with elucidation of mechanisms regulating the exocrine secretion of mucin glycoproteins by mucous cells of salivary mucous glands. As a model system for our studies we use short-term cultures of acinar structures isolated from rat sublingual glands. Recent results are discussed from an on-going study of cross-talk between the two primary signaling pathways regulating exocrine secretion from isolated sublingual acini: the muscarinic cholinergic and vasoactive intestinal peptide (VIP) pathways. The combination of muscarinic agonist (carbachol) and VIP elicits a secretion equivalent to about 150% of the additive sum of the secretory responses to each agonist alone. This synergistic secretory response is only observed at submaximal concentrations of carbachol. VIP thus serves to decrease the EC50 for carbachol nearly three-fold. Results are thus far consistent with the hypothesis that a sustained rise in the intracellular concentration of calcium ions induced by VIP accounts for synergistic secretion and the heightened sensitivity to carbachol.

Biochemical and immunological studies and assay of rat sublingual mucins.

Original studies of rat sublingual mucins raised questions as to the existence of a second mucin species as distinguished by binding to hydroxyapatite. The existence of multiple mucin species is of concern in pharmacological studies of mucous-cell secretion as each species could represent distinct mucous-cell populations that respond differently to secretagogues. Thus a separate hydroxyapatite-bound mucin pool expressed in rat sublingual glands was isolated and characterized. Biochemical comparison of hydroxyapatite-bound mucins to total and hydroxyapatite-unbound sublingual mucins demonstrated no substantial differences in either amino acid and carbohydrate contents or in size distributions. In addition, a radioimmunoassay was developed using antisera prepared previously against unbound mucins. The three mucin pools exhibited equal specificities in displacement of radiolabelled unbound mucin tracer in the radioimmunoassay. Thus, bound and unbound mucins are indistinguishable, both immunologically and in biochemical composition. The radioimmunoassay was then evaluated for use in pharmacological studies of acinar mucous-cell secretion. Measurement by radioimmunoassay of secretion from isolated acini in response to carbachol was concentration-dependent (EC50 approx. 0.3 microM and maximal stimulation at 1 microM carbachol). In immunolocalization studies the antiserum was highly selective for mucous cells, recognized all mucous cells within histological sections, and was localized subcellularly to mucous-cell secretion granules and trans-Golgi, further validating the radioimmunoassay as a method to detect exocrine secretion from the entire pool of acinar mucous cells. Moreover, the radioimmunoassay was compared and found equivalent to an acid-precipitation method to assess relative secretion, suggesting the acid-precipitation method is also valid for pharmacological studies of isolated acini.

Both M1 and M3 receptors regulate exocrine secretion by mucous acini.

We investigated the role of M1 and M3 receptors in regulating exocrine secretion from acini isolated from rat sublingual glands. In secretion experiments, we derived affinity values (KB) from Schild regression analysis for the antagonists pirenzepine (61.0 nM) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; 1.06 nM). The KB for 4-DAMP is similar to its affinity value [equilibrium dissociation constant from competition studies (Ki); 1.81 nM] determined from radioligand competition experiments. In contrast, the KB for pirenzepine is between its high-affinity (17.6 nM) and low-affinity (404 nM) Ki values. In separate secretion experiments, we found that the M1 receptor antagonist, M1-toxin, induces a rightward shift in the concentration-response curve to muscarinic agonist and inhibits maximal secretion by 40%. The inhibitory effect of M1-toxin appears specific for M1 receptor blockade, since the toxin abolishes acinar high-affinity pirenzepine-binding sites and does not inhibit secretion induced by nonmuscarinic agents. Additional pharmacological studies indicate muscarinic receptors do not function through putative neural elements within isolated acini. Our combined results are consistent with both M1 and M3 receptors directly regulating mucous acinar exocrine secretion and indicate M3 receptors alone are insufficient to induce a maximal muscarinic response.

Regulation of mucous acinar exocrine secretion with age.

Denny and co-workers (Navazesh et al., 1992) recently reported decreased concentrations of MG1 and MG2 mucins in resting and stimulated whole human saliva with age. The current study was therefore conducted to examine whether there is a corresponding attenuation with age in stimulus secretion coupling regulating mucous cell exocrine secretion. We utilized an in vitro model system, isolated rat sublingual acini, to evaluate the regulation of mucous cell exocrine secretion. Rat sublingual glands are similar to human sublingual and minor mucous glands, both histologically and in terms of their pattern of innervation, which is predominantly parasympathetic. Mucin secretion is thus activated primarily by muscarinic cholinergic agonist and to a lesser extent by vasoactive intestinal peptide (VIP), which is co-localized with acetylcholine in parasympathetic nerve terminals. We isolated sublingual mucous acini from five-month-old and 24-month-old rats and compared the concentration responses for mucin secretion induced by VIP and the muscarinic agonist, arecaidine propargyl ester (APE). Concentration-response curves for VIP were nearly identical for mucous acini from the five-month-old and 24-month-old animals. Values for basal secretion, maximal secretion, and EC50 (approximately equal to 200 nmol/L VIP) were statistically equivalent between both age groups. Concentration-response curves for APE were also very similar between age groups, with no statistically significant difference in basal secretion or EC50 values (approximately equal to 50 nmol/L APE). Maximal secretion was slightly less but statistically different for 24-month-old vs. five-month-old animals, 158% vs. 169% above basal secretion, respectively. Collectively, we found no substantial age-related changes in the secretory responsiveness of salivary mucous cells.

Persistence of a perinatal cellular phenotype in submandibular glands of adult rat.

In the perinatal submandibular gland (SMG) of the rat, Type I cells secrete protein C (89 KD) and Type III cells secrete B1-immunoreactive proteins (20-30 KD); both cell types secrete protein D (175 KD). After the disappearance of both perinatal cell types from the maturing acini, only cells of the intercalated ducts (ID) show strong reactivity for the perinatal antigens. In adult ID, light and electron microscopic immunocytochemical analysis showed that most cells had either C or B1 reactivity, a few had either C and D or B1 and D reactivities, and some cells were unreactive for all of the perinatal proteins. Occasional clusters of "adult" acini, however, were strongly positive for B1 and for D, and these clusters were negative for a typical adult acinar marker, the glutamine/glutamic acid-rich proteins (GRP). Also seen in some preparations were a few anomalous acini with the histological appearance of sublingual (SLG) acini. These were negative for the perinatal and adult submandibular gland marker proteins but reactive with an antibody against SLG mucin. We suggest that the B1-positive acini in the adult SMG consist of newly differentiated replacement cells that have arisen from the ID, and that the anomalous mucous acini are, phenotypically, SLG acini that have differentiated within the SMG parenchyma.

Composition of human airway mucins and effects after inhalation of acid aerosol.

Characterization of normal airway mucus is required to elucidate mechanisms protecting the airways and to understand changes associated with disease and environmental insult. Toward this goal, we collected bronchial washes (10 ml saline) from healthy human subjects to 1) evaluate the yield of high-density material (delta > or = 1.35 g/ml), and 2) characterize glycoconjugates associated with collected secretions. Samples were lipid extracted followed by CsCl density gradient centrifugation. The yield of high-density material from individual subjects was variable but sufficient to demonstrate that mucin glycoproteins are a major constituent of mucus from healthy airways and that proteoglycans are absent. Next, we investigated whether inhalation of H2SO4 aerosol (1,000 microgram/m3), an environmental insult associated with alterations in mucociliary clearance, changes the composition of high-density glycoproteins in airway secretions. In a paired, double-blinded study, high-density fractions of bronchial secretions from 12 subjects were collected 18 h after exposures of 2 h to aerosolized NaCl and H2SO4. In all cases the high-density material displayed characteristics of mucin glycoproteins. In addition, a unique 150-kDa glycoprotein was detected in most but not all samples and may represent a small mucin glycoprotein differentially expressed in humans. No differences were noted between the two exposure conditions in the profiles of the glycoproteins or proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Statistically, large changes with acid exposure in the composition of carbohydrates and amino acids were absent. Thus no substantial systematic changes in airway mucin glycoproteins or closely associated proteins and glycoproteins were correlated with H2SO4 exposure. Alternatively, statistical analysis of the differences between exposures in glycoprotein constituents among subjects denoted greater variability in carbohydrates compared with amino acids with repeated sampling, suggesting normal daily variations in the mucin composition of individual airway mucus.

Muscarinic cholinergic receptor subtypes in rat sublingual glands.

Mucin glycoprotein secretion by rat sublingual glands is regulated primarily by muscarinic cholinergic receptors. Studies were conducted to identify muscarinic receptor subtypes in whole glands as well as in isolated acinar structures. In radioligand binding studies, we used subtype-selective antagonists in competition studies to initially determine receptor subtype heterogeneity. In membranes from whole glands, both pirenzepine and methoctramine displayed two affinity sites (M1 and M3) of nearly equal proportions. In contrast, acinar membranes contained a 1:2 and 2:1 ratio of M1 to M3 sites for pirenzepine and methoctramine, respectively. In all cases, p-fluoro-hexahydro-siladifenidol and 4-diphenylacetoxy-N-methylpiperidine each bound to a single class of binding sites. Northern analysis using oligonucleotide probes specific for the 5' ends of the translated regions of m1 through m5 receptors detected only m1 and m3 subtypes in poly(A)+ RNA from whole glands. We also used antisera specific for each receptor subtype to immunoprecipitate solubilized receptors from membrane preparations. Only m1 (51.7 and 64.9%) and m3 (48.3 and 34.7%) subtypes were found consistently in membranes from whole sublingual glands and isolated acini, respectively. Studies with other exocrine glands generally described the predominance of m3 receptors, and m1 receptors, if present, were presumably associated with contaminating neural structures. Our results therefore demonstrate that mucous acini from rat sublingual glands contain abundant amounts of both m1 and m3 receptors.

Mucinlike glycoproteins from cat tracheal gland cells in primary culture.

In a recent study (D. J. Culp, D. K. P. Lee, D. P. Penney, and M. G. Marin. Am. J. Physiol. 263: L264-275, 1992), we reported that primary cultures of cat tracheal gland cells expressed histological, ultrastructural, and immunological characteristics of mucous cells when cultured on floating gels of rat tail collagen (released-gel cultures) compared with cells cultured on glutaraldehyde-fixed collagen gels (fixed-gel cultures). We therefore collected culture medium from gland cells grown under both culture conditions for determination and comparison of glycoconjugates with characteristics of mucin glycoproteins. Cells were cultured in the presence of [3H]glucosamine, and material of high molecular weight and density (HMD material) was isolated. HMD material from both culture conditions were each resistant to heparitinase and heparinase, whereas 72 and 25% of the radiolabel in released-gel and fixed-gel HMD material, respectively, was resistant to chondroitinase ABC. Material resistant to chondroitinase ABC was analyzed further. Both samples contained a single broad glycoprotein band [relative molecular weight (M(r)) > 250,000] after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had amino acid profiles similar to airway mucin. The sample from fixed-gel cultures had nearly equal amounts of carbohydrate and protein, was highly enriched in N-acetylglucosamine, contained mannose, displayed little blood group A immunoreactivity, and had few O-linked oligosaccharides. Conversely, the sample from released-gel cultures contained 80% carbohydrate, was composed of monosaccharides characteristic of airway mucins, displayed blood group A immunoreactivity, and contained oligosaccharides O-linked via N-acetylgalactosamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Sulfuric acid aerosol exposure in humans assessed by bronchoalveolar lavage.

Epidemiologic and experimental evidence suggests that exposure to acidic aerosols may affect human health. Brief exposures to acidic aerosols alter mucociliary clearance and increase airway responsiveness, but effects on host defense mechanisms at the alveolar level have not been studied in humans. Twelve healthy, nonsmoking volunteers between 20 and 39 yr of age were exposed for 2 h to aerosols of approximately 1,000 micrograms/m3 sulfuric acid (H2SO4) or sodium chloride (NaCl [control]), with intermittent exercise, in a randomized, double-blind fashion. Each subject received both exposures, separated by at least 2 wk. Bronchoalveolar lavage (BAL) was performed 18 h after exposure in order to detect evidence of an inflammatory response, changes in alveolar cell subpopulations, or changes in alveolar macrophage (AM) function, which is important in host defense. When compared with NaCl, exposure to H2SO4 did not increase polymorphonuclear leukocytes in BAL fluid. The percentage of T lymphocytes decreased in association with H2SO4 exposure, but the difference was not statistically significant (14.9% after NaCl, 11.5% after H2SO4; p = 0.14). Antibody-mediated cytotoxicity of AM increased in association with H2SO4 exposure (percent lysis 19.1 after NaCl, 23.6 after H2SO4; p = 0.16). No significant change was seen in release of superoxide anion or inactivation of influenza virus in vitro. Brief exposures to H2SO4 aerosol at 1,000 micrograms/m3 do not cause an influx of inflammatory cells into the alveolar space, and no evidence was found for alteration in antimicrobial defense 18 h after exposure.

Cat tracheal gland cells in primary culture.

Conditions for the primary culture of isolated cat tracheal gland cells were established. Cells plated onto glutaraldehyde-fixed gels of rat tail collagen grew to confluency after 8 days of culture forming a monolayer of cuboidal cells with ultrastructural characteristics of epithelial cells and immunoreactivity to antikeratins. Cultured cells synthesized and released radiolabeled high-molecular-weight glycoconjugates. Glycoconjugate secretion was increased approximately 10% in response to the cholinergic agonist, carbachol. Secretion of glycoconjugates was unrelated to regulated exocrine secretion, since these cells were devoid of secretion granules as assessed by light and electron microscopy. Confluent cultures also generated a spontaneous potential difference and short-circuit current, which were both inhibited by ouabain and increased by carbachol. This suggested gland cells contribute to fluid secretion by active ion-transport mechanisms. We also plated cells onto unfixed collagen gels that were released from the culture dish at confluency. Cells were columnar with apically oriented secretion granules that stained with alcian blue and for blood group A immunoreactivity. Secretion of radiolabeled high-molecular-weight glycoconjugates was increased 27% by carbachol. These cell culture systems may serve as models to investigate glandular secretory mechanisms and their regulation.

Rat sublingual gland as a model to study glandular mucous cell secretion.

To study the regulation of mucous cell secretion, we have developed an in vitro cell model consisting of enzymatically dispersed mucous acinar structures (cell aggregates) from rat sublingual glands. Histological and ultrastructural evidence demonstrates that the cell aggregates are highly enriched in mucous cells, retain the morphological and ultrastructural features observed in intact glands, and undergo transition to an extensive secretory state when stimulated by 10 microM carbachol. The secretory responsiveness of the cell aggregates was verified in pharmacological studies. Carbachol stimulated secretion in a dose-dependent manner with high affinity (concentration causing half-maximal response = 0.3 microM) and was completely inhibited by atropine. Secretion was also stimulated by vasoactive intestinal peptide and substance P but not by alpha- or beta-adrenergic agonists. Biochemical characterization of secretion during nonstimulated and carbachol-stimulated conditions (after preincubation in [3H]glucosamine) demonstrated that, in response to carbachol, cell aggregates synthesized and secreted mucins which were similar to mucin glycoproteins isolated from whole glands. Collectively, our results establish that the rat sublingual cell aggregate model is a viable and pharmacologically responsive cell system to study the regulation of mucous cell secretion.

Variable effects of soman on macromolecular secretion by ferret trachea.

The purpose of this study was to examine the effect of the anticholinesterase agent, soman, on macromolecular secretion by ferret trachea, in vitro. We mounted pieces of ferret trachea in Ussing-type chambers. Secreted sulfated macromolecules were radiolabeled by adding 500 microCi of 35SO4 to the submucosal medium and incubating for 17 hr. Soman added to the submucosal side produced a concentration-dependent increase in radiolabeled macromolecular release with a maximal secretory response (mean +/- SD) of 202 +/- 125% (n = 8) relative to the basal secretion rate at a concentration of 10(-7) M. The addition of either 10(-6) M pralidoxime (acetylcholinesterase reactivator) or 10(-6) M atropine blocked the response to 10(-7) M soman. At soman concentrations greater than 10(-7) M, secretion rate decreased and was not significantly different from basal secretion. Additional experiments utilizing acetylcholine and the acetylcholinesterase inhibitor, physostigmine, suggest that inhibition of secretion by high concentrations of soman may be due to a secondary antagonistic effect of soman on muscarinic receptors.

Alpha-adrenergic regulation of secretion by tracheal glands.

The purpose of the present study was to begin to characterize, pharmacologically, the alpha-adrenergic regulation of glycoconjugate secretion from airway glands. Using isolated gland cells from cat trachea, we determined the binding characteristics of [3H]dihydroergocryptine ([3H]DHE), an alpha-adrenergic antagonist, with equal affinities for alpha 1- and alpha 2-adrenergic receptors. Specific binding of [3H]DHE to gland cell homogenates was saturable, of high affinity (KDapp = 4.2 nM) and inhibited with greater efficacy by epinephrine much greater than isoproterenol. Competition experiments with alpha 1- and alpha 2-adrenergic selective antagonists (prazosin and yohimbine, respectively) demonstrated high- and low-affinity sites for each antagonist, indicating the presence of both receptor subtypes. In studies of glycoconjugate secretion by cat tracheal explants, secretion was stimulated by adrenergic agonists with the rank potency: norepinephrine greater than or equal to phenylephrine greater than epinephrine much greater than clonidine. alpha-Adrenergic-stimulated secretion (epinephrine + propranolol) was inhibited by low concentrations of prazosin, but was unaffected by 100 nM yohimbine. The alpha 2-adrenergic agonists, clonidine and UK-14,304, each markedly inhibited beta-adrenergic-stimulated secretion. Collectively, these results demonstrate alpha 1-adrenergic regulation of glandular glycoconjugate secretion and suggest alpha 2-adrenergic receptors may modulate beta-adrenergic-stimulated secretion.