The leishmanial lipophosphoglycan: a multifunctional molecule

A striking characteristic of leishmanial parasites is their ability to avoid destruction in hostile environments throughout their life cycle. To survive, the parasites must have evolved specialized molecules. One such molecule that has received considerable attention is an unusual glycoconjugate called lipophosphoglycan. The macromolecule is the major cell surface glycoconjugate of all Leishmania promastigotes. This minireview summarizes current information on the structure and possible functions of this intriguing molecule.     

Histochemistry of glycoconjugates in mucous cells of Salmo trutta uninfected and naturally parasitized with intestinal helminths

Mucus secreted onto the surface of the intestine forms a physical barrier to invading parasites so that a possible attachment of helminths to the surface is prevented and their expulsion by peristalsis facilitated. In mammals, intestinal parasites induce hyperplasia and hypertrophy of intestine goblet cells and provoke changes in the mucus composition. In fish, this topic has received less attention. In the present investigation, histochemical methods were employed to compose intestinal mucous cell numbers and their glycoconjugate composition were compared by uninfected brown trout Salmo trutta and in S. trutta parasitized with Cyathocephalus truncatus or Pomphorhynchus laevis. When P. laevis was present in the intestine of the brown trout, the total mucous cell number, and the number of mucous cells containing acid or mixed glycoconjugates were significantly enhanced. No significant change in the total mucous cell number was detected in the intestine of fish parasitized with C. truncatus in comparison with uninfected brown trout. A significant increase was observed in the number of both acid (especially sulphated) and mixed glycoconjugates containing mucous cells as well as a significant decrease in the number of neutral glycoconjugates containing mucous cells. When intestinal helminths were present, the thickness of the adherent mucous gel increased. In a limited number of other fish species, the occurrence of gill and intestinal parasites has been reported to increase the mucosal glycoconjugate secretions. Our study is the first quantitative report on the effects of intestinal helminths on the density of mucous cells and mucus composition in a fish species.     

Requirement of lipophosphoglycan for intracellular survival of Leishmania donovani within human monocytes

The function of the lipophosphoglycan of Leishmania donovani parasites was investigated in human peripheral monocytes. In contrast to wild-type L. donovani which grow in monocytes, incubation of monocytes with two variant lines of L. donovani, defective in lipophosphoglycan expression, resulted in the entry of the variant cells into the monocytes and their subsequent destruction. Passive transfer of lipophosphoglycan to the variant cells led to prolonged survival in monocytes. These results indicate that lipophosphoglycan is required by the parasite for intracellular survival. To investigate one possible protective role of the glycoconjugate, preincubation of monocytes with a suspension of lipophosphoglycan and subsequent treatment of the cells with PMA or opsonized zymosan resulted in an attenuation of the oxidative burst; the attenuation effect was concentration dependent on the glycoconjugate and independent of preincubation time. Moreover, hydrophobic beads, coated with lipophosphoglycan, were phagocytized by monocytes and found to inhibit oxygen consumption in monocytes activated with PMA. These results suggest a possible relationship between the absence of lipophosphoglycan in the variant parasites and their inability to survive within monocytes. Although the precise molecular basis remains to be elucidated, the ability of lipophosphoglycan to impair the microbial oxidative response may be a contributing factor in its requirement for intracellular survival.     

An amphipathic sulphated glycoconjugate of Leishmania: characterization with monoclonal antibodies

A major glycoconjugate of Leishmania tropica major identified by two monoclonal antibodies was shown to be an externally oriented, amphipathic membrane antigen shed into the culture medium in which the parasites grow. This molecule could be labelled metabolically with [3H]glucose, [3H]galactose, [32P]phosphate and [35S]sulphate. It migrated as a polydisperse band upon electrophoresis in SDS-polyacrylamide gels, spanning the region of the gel corresponding to an apparent mol. wt. of 20 000-67 000 daltons. An apparently identical family of molecules could be labelled on the surface of living promastigotes using galactose oxidase and [3H]-sodium borohydride. This molecule was shown to be released into the supernatant over a period of several hours. Detection of the 3H- or 35S-labelled molecule required several days exposure of autoradiographs, but a novel blotting technique using nitrocellulose coated with monoclonal antibody allowed rapid detection of the molecule in charge shift electrophoresis, Western blotting and dot blotting. The electrophoretic mobility of the glycoconjugate in agarose relative to its mobility in Triton X-100 was increased in the presence of deoxycholate, and decreased in the presence of cetyl trimethyl-ammonium bromide, indicating amphipathic properties consistent with insertion into the lipid bilayer of the membrane. Using the dot-blotting technique the glycoconjugate was detected in all virulent and avirulent clones of LRC-L137 and in two additional isolates of L. tropica major (LRC-L287 and LRC-L251), but not in L. donovani or L. mexicana, consistent with the previously described specificity of the antibodies. However, the general approaches used in this paper showed that L. donovani (LRC-L52) and L. mexicana (LRC-L94) synthesize a similar, but antigenically distinct glycoconjugate.     

Phenotypic changes associated with deletion and overexpression of a stage-regulated gene family in Leishmania

The LmcDNA16 locus of Leishmania major contains three highly related genes HASPA1 , HASPA2 and HASPB , encoding hydrophilic, acylated surface proteins and a tandem pair of unrelated sequences, SHERP1 and SHERP2 , coding for a small, hydrophilic protein that localizes to the endoplasmic reticulum and outer mitochondrial membrane. Differential regulation of these genes results in expression of a subset of the HASP proteins and SHERP only in infective stage parasites. To assess the contribution of these molecules to parasite virulence, the diploid LmcDNA16 gene locus has been removed by targeted gene deletion. Homozygous null mutants have precise deletions of both alleles and exhibit no HASP or SHERP expression. They are at least as virulent as wild-type parasites in macrophage invasion and intracellular survival assays, both in vitro and in vivo . Conversely, null mutants engineered to overexpress the entire LmcDNA16 gene locus are unable to survive within the intramacrophage environment despite their differentiation into infective metacyclic parasites. Both null and overexpressing null parasites show increased sensitivity to complement-mediated lysis, suggesting perturbation of their surface architecture. Avirulence in overexpressing parasites correlates with selective depletion of a specific lipid species, decreased expression of the major surface glycoprotein GP63, but no significant downregulation of the glycoconjugate lipophosphoglycan.     

The role of the NTPDase enzyme family in parasites: what do we know, and where to from here?

Nucleoside triphosphate diphosphohydrolases (NTPDases, GDA1_CD39 protein superfamily) play a diverse range of roles in a number of eukaryotic organisms. In humans NTPDases function in regulating the inflammatory and immune responses, control of vascular haemostasis and purine salvage. In yeast NTPDases are thought to function primarily in the Golgi, crucially involved in nucleotide sugar transport into the Golgi apparatus and subsequent protein glycosylation. Although rare in bacteria, in Legionella pneumophila secreted NTPDases function as virulence factors. In the last 2 decades it has become clear that a large number of parasites encode putative NTPDases, and the functions of a number of these have been investigated. In this review, the available evidence for NTPDases in parasites and the role of these NTPDases is summarized and discussed. Furthermore, the processes by which NTPDases could function in pathogenesis, purine salvage, thromboregulation, inflammation and glycoconjugate formation are considered, and the data supporting such putative roles reviewed. Potential future research directions to further clarify the role and importance of NTPDases in parasites are proposed. An attempt is also made to clarify the nomenclature used in the parasite field for the GDA1_CD39 protein superfamily, and a uniform system suggested.     

Saccharide anions as inhibitors of the malaria parasite

The asexual erythrocytic stage of Plasmodium falciparum was grown in culture in the presence or absence of glycoconjugate polyanions of varying structure, size and substitutions. Heparin, dextran sulfate, fucoidan and pentosan polysulfate had antimalarial IC50 values between one and 11 μg ml−1. Constituent heparin disaccharides were ineffective against the malaria parasite and desulfation from either the O- or N-substitution sites of heparin or reduction of the uronic acid carboxyl group neutralized the antimalarial response to varying degrees. Immobilization of heparin onto agarose beads still permitted antimalarial activity suggesting that parasite uptake of the glycoconjugate is not required for inhibition. Accordingly, it is concluded that invasion of free parasites into the erythrocytes was inhibited rather than parasite maturation within the red cell. Merozoite surface antigen-1 was apparently prevented from binding to human erythrocytes in the presence of highly sulfated polyanions and, in a dose-dependent fashion, heparin. Abbreviations: MSA-1, merozoite surface antigen-1 This revised version was published online in November 2006 with corrections to the Cover Date.     

Galectin-1 on cervical epithelial cells is a receptor for the sexually transmitted human parasite Trichomonas vaginalis

The extracellular protozoan parasite Trichomonas vaginalis causes the most prevalent non-viral sexually transmitted human infection, yet the pathogenesis of infection is poorly understood, and host cell receptors have not been described. The surface of T. vaginalis is covered with a glycoconjugate called lipophosphoglycan (LPG), which plays a role in the adherence and cytotoxicity of parasites to human cells. T. vaginalis LPG contains high amounts of galactose, making this polysaccharide a candidate for recognition by the galactose-binding galectin family of lectins. Here we show that galectin-1 (gal-1) is expressed by human cervical epithelial cells and binds T. vaginalis LPG. Gal-1 binds to parasites in a carbohydrate-dependent manner that is inhibited in the presence of T. vaginalis LPG. Addition of purified gal-1 to cervical epithelial cells also enhances parasite binding, while a decrease in gal-1 expression by small interfering RNA (siRNA) transfection decreases parasite binding. In contrast, the related galectin-7 (gal-7) does not bind T. vaginalis in a carbohydrate-dependent manner, and is unable to mediate attachment of parasites to host cells. Our data are consistent with the presence of multiple host cell receptors for T. vaginalis of which gal-1 is the first to be identified and highlight the importance of glycoconjugates in host-pathogen interactions.     

Refined structure of the lipophosphoglycan of Leishmania donovani.

The primary structure of the major surface glycoconjugate of Leishmania donovani parasites, a lipophosphoglycan, has been further characterized. The repeating PO4-6Galp beta 1-4Man disaccharide units, which are a salient feature of the molecule, are shown to terminate with one of several neutral structures, the most abundant of which is the branched trisaccharide Galp beta 1-4(Manp alpha 1-2)Man. The phosphosaccharide core of lipophosphoglycan, which links the disaccharide repeats to a lipid anchor, contains 2 phosphate residues. One of the core phosphates has previously been localized on O-6 of the galactosyl residue distal to the lipid anchor; the second phosphate is now shown to be on O-6 of the mannosyl residue distal to the anchor and to bear an alpha-linked glucopyranosyl residue. Also, the anomeric configuration of the unusual 3-substituted Galf residue in the phosphosaccharide core is established as beta. The complete structure of the core is thus PO4-6Galp alpha 1-6Galp alpha 1-3Galf beta 1-3[Glcp alpha 1-PO4-6]Manp alpha 1-3Manp alpha 1-4GlcN alpha 1-. This further clarification of the structure of lipophosphoglycan may prove beneficial in determining the structure-function relationships of this highly unusual glycoconjugate.     

Species-specific antibodies to Leishmania tropica (minor) recognize somatic antigens and exometabolites

Monoclonal antibodies were raised against membranes of Leishmania tropica (formerly L. tropica minor). These antibodies do not react with membranes or homogenates prepared from any other species of Leishmania examined. The antigens identified by all four monoclonal antibodies reside on the external surface of the promastigote and are released into the culture medium. Immunoprecipitation of metabolically ([3H]glucose, -glucosamine, or [35S]methionine)-labeled, [125I]lactoperoxidase-labeled, or Western blot analysis of the parasites identified species-specific determinants residing on several different molecules with Mr between 12,000 and 140,000. All of the monoclonal antibodies appear to recognize determinants on a common [3H]sugar-labeled glycoconjugate, Mr = 32,000 to 44,000, but also react with additional components of 55,000, 80,000, 92,000, and 130,000 m.w. Antibody binding to the parasites is very sensitive to mild oxidation with periodate, suggesting that the species-specific determinants are carbohydrate in nature.     

Isolation of a high molecular weight glycoconjugate derived from the surface of S purpuratus eggs that is implicated in sperm adhesion

Sea urchin sperm–egg adhesion is mediated by bindin, a sperm surface protein that has lectin-like activity. Bindin agglutinates eggs, and this interaction has been shown to be inhibited by glycopeptides released from the egg surface by protease treatment. In this study, we report the purification and properties of such an egg surface glycoconjugate that may be involved in sperm adhesion. The glycoconjugate was partially purified by gel filtration and affinity chromatography on bindin particles. Upon gel filtration on Sepharose CL 4-B, the glycoconjugate elutes near the void volume, suggesting that it has a molecular weight in excess of one million. In addition, we have found that the egg surface glycoconjugate agglutinates bindin particles, indicating that it is multivalent. Carbohydrate analysis indicates that the glycoconjugate is composed primarily of fucosc, xylose, galactose, and glucose. This purified egg surface component is the most potent inhibitor of bindin-mediated egg agglutination yet described.     

A glycoconjugate from corms of saffron plant (Crocus sativus L.) inhibits root growth and affects in vitro cell viability

A glycoconjugate has been characterized from saffron corms (Crocus sativus L.) that inhibits the growth of roots of Nicotiana tabacum and Arabidopsis thaliana, at concentrations ranging from 1-100 micrograms m-3. Roots of seedlings grown in the presence 0.1 microgram m-3 glycoconjugate showed bulging of epidermal cells, whereas at 10 micrograms m-3, roots were completely devoid of hairs. At 100 micrograms m-3 glycoconjugate the cell walls of the root vascular tissues were thicker and, overall, the vascular tissue was enlarged. In addition, this glycan is cytotoxic to isolated tobacco cells and protoplasts, with 50% cell death induced by 0.5 and 2 micrograms m-3 glycoconjugate, respectively. Morphological and biochemical changes induced by the exposure to the glycoconjugate included cell size decrease, loss of regular cell shape, cytoplasm collapse, and release of intracellular proteins. This molecule at low concentrations (0.1 microgram m-3) mimics the effects of Yariv phenylglycosides and of mutant Arabidopsis which present defective or missing arabinogalactan-proteins (AGPs) in roots, indicating the glycoconjugate might interact with cell surface AGPs.     

Impaired recruitment of the small GTPase rab7 correlates with the inhibition of phagosome maturation by Leishmania donovani promastigotes

We have shown recently that one of the survival strategies used by Leishmania donovani promastigotes during the establishment of infection in macrophages consists in inhibiting phagosome–endosome fusion. This inhibition requires the expression of lipophosphoglycan (LPG), the predominant surface glycoconjugate of promastigotes, as parasites expressing truncated forms of LPG reside in phagosomes that fuse extensively with endocytic organelles. In the present study, we developed a single-organelle fluorescence analysis approach to study and analyse the intracellular trafficking of 'fusogenic' and 'low-fusogenic' phagosomes induced by an LPG repeating unit-defective mutant ( lpg2 KO) or by wild-type L. donovani promastigotes respectively. The results obtained indicate that phagosomes containing mutant parasites fuse extensively with endocytic organelles and transform into phagolysosomes by losing the early endosome markers EEA1 and transferrin receptor, and acquiring the late endocytic and lysosomal markers rab7 and LAMP1. In contrast, a majority of 'low-fusogenic' phagosomes containing wild-type L. donovani promastigotes do not acquire rab7, wheres they acquire LAMP1 with slower kinetics. These results suggest that L. donovani parasites use LPG to restrict phagosome–endosome fusion at the onset of infection in order to prevent phagosome maturation. This is likely to permit the transformation of hydrolase-sensitive promastigotes into hydrolase-resistant amastigotes within a hospitable vacuole not displaying the harsh environment of phagolysosomes.     

Disruption of mannose activation in Leishmania mexicana: GDP-mannose pyrophosphorylase is required for virulence, but not for viability.

In eukaryotes, the enzyme GDP-mannose pyrophosphorylase (GDPMP) is essential for the formation of GDP-mannose, the central activated mannose donor in glycosylation reactions. Deletion of its gene is lethal in fungi, most likely as a consequence of disrupted glycoconjugate biosynthesis. Furthermore, absence of GDPMP enzyme activity and the expected loss of all mannose-containing glycoconjugates have so far not been observed in any eukaryotic organism. In this study we have cloned and characterized the gene encoding GDPMP from the eukaryotic protozoan parasite Leishmania mexicana. We report the generation of GDPMP gene deletion mutants of this human pathogen that are devoid of detectable GDPMP activity and completely lack mannose-containing glycoproteins and glycolipids, such as lipophosphoglycan, proteophosphoglycans, glycosylphosphatidylinositol protein membrane anchors, glycoinositolphospholipids and N-glycans. The loss of GDPMP renders the parasites unable to infect macrophages or mice, while gene addback restores virulence. Our study demonstrates that GDP-mannose biosynthesis is not essential for Leishmania viability in culture, but constitutes a virulence pathway in these human pathogens.     

The role of phosphomannose isomerase in Leishmania mexicana glycoconjugate synthesis and virulence

Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of fructose 6-phosphate and mannose 6-phosphate, which is the first step in the biosynthesis of activated mannose donors required for the biosynthesis of various glycoconjugates. Leishmania species synthesize copious amounts of mannose-containing glycolipids and glycoproteins, which are involved in virulence of these parasitic protozoa. To investigate the role of PMI for parasite glycoconjugate synthesis, we have cloned the PMI gene (lmexpmi) from Leishmania mexicana, generated gene deletion mutants (Delta lmexpmi), and analyzed their phenotype. Delta lmexpmi mutants lack completely the high PMI activity found in wild type parasites, but are, in contrast to fungi, able to grow in media deficient for free mannose. The mutants are unable to synthesize phosphoglycan repeats [-6-Gal beta 1-4Man alpha 1-PO(4)-] and mannose-containing glycoinositolphospholipids, and the surface expression of the glycosylphosphatidylinositol-anchored dominant surface glycoprotein leishmanolysin is strongly decreased, unless the parasite growth medium is supplemented with mannose. The Delta lmexpmi mutant is attenuated in infections of macrophages in vitro and of mice, suggesting that PMI may be a target for anti-Leishmania drug development. L. mexicana Delta lmexpmi provides the first conditional mannose-controlled system for parasite glycoconjugate assembly with potential applications for the investigation of their biosynthesis, intracellular sorting, and function.     

Identification of versican as an isolectin B4-binding glycoprotein from mammalian spinal cord tissue

Nociceptors are specialized nerve fibers that transmit noxious pain stimuli to the dorsal horn of the spinal cord. A subset of nociceptors, the nonpeptidergic C-fibers, is characterized by its reactivity for the plant isolectin B4 (IB4) from Griffonia simplicifolia. The molecular nature of the IB4-reactive glycoconjugate, although used as a neuroanatomical marker for more than a decade, has remained unknown. We here present data which strongly suggest that a splice variant of the extracellular matrix proteoglycan versican is the IB4-reactive glycoconjugate associated with these nociceptors. We isolated (by subcellular fractionation and IB4 affinity chromatography) a glycoconjugate from porcine spinal cord tissue that migrated in SDS/PAGE as a single distinct protein band at an apparent molecular mass of > 250 kDa. By using MALDI-TOF/TOF MS, we identified this glycoconjugate unambiguously as a V2-like variant of versican. Moreover, we demonstrate that the IB4-reactive glycoconjugate and the versican variant can be co-released from spinal cord membranes by hyaluronidase, and that the IB4-reactive glycoconjugate and the versican variant can be co-precipitated by an anti-versican immunoglobulin and perfectly co-migrate in SDS/PAGE. Our findings shed new light on the role of the extracellular matrix, which is thought to be involved in plastic changes underlying pain-related phenomena such as hyperalgesia and allodynia.     

Quantitation of Leishmania lipophosphoglycan repeat units by capillary electrophoresis

The glycosylphosphatidylinositol (GPI)-anchored lipophosphoglycan (LPG) of Leishmania is the dominant cell surface glycoconjugate of these pathogenic parasites. LPG is structurally characterized by a series of phosphoglycan repeat units. Determining the number of repeat units per LPG molecule has proven difficult using current technologies, such as mass spectrometry. As an alternative method to quantitate the number of repeat units in LPG, a procedure based on capillary electrophoretic analysis of the proportion of mannose to 2,5-anhydromannose (derived from the nonacetylated glucosamine of the GPI anchor of LPG) was developed. The CE-based technique is sensitive and relatively rapid compared to GC-MS-based protocols. Its application was demonstrated in quantitating the number of LPG repeat units from several species of Leishmania as well as from two life-cycle stages of these organisms.     

Cell-free biosynthesis of lipophosphoglycan from Leishmania donovani. Characterization of microsomal galactosyltransferase and mannosyltransferase activities

Incubation of microsomal preparations from Leishmania donovani parasites with UDP-[3H]galactose or GDP-[14C]mannose resulted in incorporation of radiolabel into an endogenous product that exhibited the chemical and chromatographic characteristics of the parasite's major surface glycoconjugate, lipophosphoglycan. The [3H]galactose- or [14C]mannose-labeled product was (i) cleaved by phosphatidylinositol-specific phospholipase C; (ii) deaminated by nitrous acid; and (iii) degraded into radioactive, low molecular weight fragments upon hydrolysis with mild acid. Analysis of the products of mild acid hydrolysis revealed the presence of phosphorylated Gal-beta-Man as the major fragment with lesser amounts of mono-, tri-, and tetrasaccharides. The incorporation of the two isotopic precursors was neither stimulated by the addition of dolichylphosphate nor inhibited by amphomycin, indicating that dolichol-saccharide intermediates are not involved in assembly of the repeating units of lipophosphoglycan. Development of this cell-free glycosylating system will facilitate further studies on the pathway and enzymes involved in lipophosphoglycan biosynthesis.     

Transmembrane assemblage of the photoreceptor connecting cilium and motile cilium transition zone contain a common immunologic epitope.

The photoreceptor connecting cilium bears a unique transmembrane assemblage which stably links cell surface glycoconjugates with the underlying axonemal cytoskeleton. Structural similarities between the photoreceptor connecting cilium and the transition zone of motile cilia suggests that this assemblage may also be present in motile cilia. Using a subcellular fraction enriched in detergent-extracted photoreceptor axonemes, three high molecular mass glycoconjugates (425, 600, and 700 kD) were previously identified as potential components of the assemblage. Through oligosaccharide characterization and binding of a specific monoclonal antibody, we have verified the localization of the 425 kD glycoconjugate to the transmembrane assemblage. Binding of the lectin peanut agglutinin (PNA) to the 425 kD glycoconjugate on nitrocellulose blots, and to isolated detergent-extracted axonemes, was assessed following treatment with the enzymes neuraminidase and O-glycanase. Changes in binding to the 425 kD glycoconjugate precisely paralleled changes in binding to intact axonemes, supporting the hypothesis that the 425 kD glycoconjugate is a component of the transmembrane assemblage. Furthermore, the results suggest that the 425 kD glycoconjugate contains sialated galactose-N-acetylgalactosamine oligosaccharides which are O-linked to the protein backbone. To directly assess the distribution of the 425 kD glycoconjugate, we produced a monoclonal antibody directed against this glycoconjugate. The antibody, K26, recognizes only the 425 kD on transblots of the axoneme fraction. K26 immunoreactivity of intact axonemes is identical to that seen by PNA staining. K26 staining of isolated photoreceptors and whole retina is uniquely localized to the region of the connecting cilium. Thus, in the photoreceptor, the 425 kD is not only a component of the transmembrane assemblage but is also completely restricted to the connecting cilium. Based on morphological similarities, the photoreceptor connecting cilium is thought to be homologous to the transition zone of the motile cilium. As such, we have stained oviduct epithelium with the K26 monoclonal antibody. Immunoreactivity is restricted to the region of the transition zone at the base of motile cilia.     

Glycoconjugate secretion in human airways in vitro: effects of epithelium removal.

The aim of this study was to examine glycoconjugate secretion in human airways with and without an epithelium. Glycoconjugate release in supernatants derived from human airways in vitro was determined using an ELISA assay with an anti-human mucin monoclonal antibody (MAb 3D3). This monoclonal antibody reacted strongly with Le(b) antigen but also recognized in vitro Le(a) and Le(y) determinants. In 11 of the 34 different lung samples (32%) studied the glycoconjugate levels were below the threshhold of detection for this assay. The mean basal secretion of glycoconjugates in human airways in vitro was 100+/-28 microg/g tissue (Period I; n = 23 different lung samples). The amount of glycoconjugate measured in the medium derived from human isolated bronchial ring preparations did not change under control conditions during the course of the experimental procedure (Period I; 128+/-46 microg/g tissue and Period II; 159 +/-48 microg/g tissue; n = 13 paired lung samples). In the supernatants of airway preparations with an intact epithelium the amount of glycoconjugates detected was 90+/-38 microg/g tissue (Period I; n = 12 different lung samples) and removal of the epithelium did not alter this basal glycoconjugate release (94+/-60 microg/g tissue: Period I, n = 8 different lung samples). The absence of the epithelial layer was confirmed by histological evaluation. Methacholine (100 microM) induced a 10- and four-fold increase in glycoconjugate release from airways with and without an epithelium, respectively. In contrast, in preparations with an epithelium, LTD4 (10 microM) and anti-IgE (dilution: 1/1000) did not cause an increase of glycoconjugate release. The methacholine difference between airways with and without an epithelium was not significantly different (P > 0.10). However, a treatment with atropine (100 microM) prevented the increase of glycoconjugate release in preparations with an epithelium. These data derived from a limited number of experiments suggest that the epithelium may not regulate the basal or stimulated release of glycoconjugates from isolated human airways.