Accumulation and localization of two adult acinar cell secretory proteins during development of the rat submandibular gland.

The seromucous acinar cells of the adult rat submandibular gland secrete a characteristic mucin glycoprotein and a family of unusual glutamine/glutamic acid-rich proteins (GRP). Monoclonal antibodies to the mucin and GRP localized in a very few Type III cells in glands of newborn and 1 day-old rats, using light and electron microscopic immunocytochemistry. Both mucin and GRP reactivities were present in the polymorphic Type IIIP granules during the 1st postnatal week. By 9 days after birth, the granules contained both mucin and GRP and were mucous-like in appearance. At earlier stages, however, cells containing only GRP or mucin could be found, indicating that the initiation of GRP and mucin biosynthesis may not be coordinately regulated. No reactivity was seen in the neonatal Type I cells or in duct cells at any age. Northern and Western blot analysis showed GRP mRNA and protein levels to be barely detectable at birth, with marked increases during the first 2 postnatal weeks. In contrast, Western blots of B1-immunoreactive proteins (B1-IP) showed levels highest in the 1st week and markedly decreased in the adult. Immunocytochemical colocalization, using gold particles of different sizes, showed that the B1-IP, mucin, and GRP colocalized in the granules. These results strengthen the hypothesis that the adult acinar cells develop from the neonatal Type III cells. No evidence was obtained for the involvement of Type I cells in the pathway of acinar cell development.     

Immunogold localization of the type II regulatory subunit of cyclic AMP-dependent protein kinase. Monoclonal antibody characterization and RII distribution in rat parotid cells

A mouse monoclonal antibody of the IgM class, MAb BB1, specific for the type II regulatory subunit (RII) of cyclic AMP-dependent protein kinase (cAPK), was produced using a purified subcellular protein fraction from rat parotid gland as the original antigen. The antibody immunoprecipitated radioactivity labeled RII from bovine heart cAPK, and from rat and human parotid saliva. Western blot analysis revealed specific binding of the antibody to proteins of 52 and 54 KD in extracts of rat parotid tissue, parotid saliva, and bovine heart cAPK. Immunogold labeling of thin sections of rat parotid gland revealed specific labeling of acinar cell nuclei (especially the heterochromatin), cytoplasm (particularly in areas containing granular endoplasmic reticulum), and the content of secretory granules. Labeling was greatly reduced (approximately 84%) when the antibody was pre-absorbed with an excess of bovine heart cAPK. In duct cells the cytoplasm and nuclei were also labeled, but few gold particles were present over secretory granules. These results provide additional evidence for the presence of nuclear cAPK in rat parotid cells, and confirm previous observations on the presence of cAPK regulatory subunits in acinar secretory granules and saliva. The hybridoma reagent will be used for studies of stimulus responses in the parotid and for immunocytochemical analyses of RII distribution in other secretory tissues.     

Ultrastructural aspects of cat submandibular glands

Submandibular glands of five adult female cats were examined by conventional electron microscopic techniques. All gland acini are mucous secreting and each acinus is capped with mucous secreting demilunar cells. Secretory product of demilunar cells is more electron lucent than that of acinar cells. The demilunes show intercellular tissue spaces and intercellular canaliculi whereas similar specializations are absent between acinar cells. Mitochondria and arrays of granular endoplasmic reticulum are more numerous in demilunar cells than in acinar cells. In acinar and demilunar cells secretory droplets first appear as enlarged Golgi saccules which subsequently become closely related to cisternae of the granular endoplasmic reticulum. Filamentous structures, interpreted as mucin molecules, are present in secretory droplets of acinar cells. Intercalated ducts are short, consisting of several junctional cells between acini and striated ducts. Striated ducts are long and tortuous and contain light cells, dark cells and basal cells. Light cells contain numerous membrane bound granules in their distal ends whereas dark cells show electron lucent vesicles in the same position. Basal cells contain a paucity of organelles and membrane plications but exhibit hemidesmosomes along their basal plasma membranes. Myoepithelial cells are abundant in relation to acinar and demilunar cells. Nerve terminals are present in some instances between acinar cells or between acinar and myoepithelial cells.     

Heterogeneity of glycoconjugates in the secretory cells of the chinchilla middle ear and eustachian tubal epithelia: a lectin-gold cytochemical study

The present study was conducted to characterize and localize the glycoconjugates in the tubotympanum (auditory or eustachian tube and middle ear cavity) of chinchilla on an ultrastructural level, using lectin-gold complexes with six different lectins: BPA, ConA, RCA-1, WGA, LFA, and SNA. A comparison of the affinity of these lectins demonstrated the heterogeneity of secretory cells. The glandular serous cells and epithelial dark granulated cells produced "serum"-type glycoprotein. The glandular mucous cells and goblet cells produced dominantly "mucin"-type glycoprotein in the light granules, but "serum"-type glycoprotein in the dark cores. The labeling of LFA and SNA showed that sialic acids existed mainly in the mucinous granules of secretory cells and ciliated epithelium glycocalyx, and in the mucous blanket. The results also suggested that the dominant linkage of sialic acids of mucin is a Neu5Ac(alpha 2-6)Gal/GalNAc sequence. Furthermore, the data obtained from ConA and BPA suggested that initial O-glycosylation of mucin took place in the cis side of the Golgi apparatus and that initial N-glycosylation of the serum occurred in the rough endoplasmic reticulum.     

Mucin histochemistry of submandibular and parotid salivary glands of man: light and electron microscopy

Light-microscopy showed parotid serous acinar cells to contain neutral mucin, serous and mucous acinar cells of submandibular gland and intercalary ductal cells of both glands to contain acid and neutral mucins, and cells of striated ducts and excretory ducts to contain neutral mucin. Mucins were demonstrated ultrastructurally in a portion of the components of secretory granules of acinar cells and intercalary ductal cells, and in secretory granules of striated and excretory ductal cells. The mucins were all stained by techniques that reveal 1,2-glycols. Secretory granules of submandibular mucous and serous acinar cells and intercalary ductal cells were stained variably by the low iron-diamine technique for acid mucin, and those of mucous acinar cells by the high iron-diamine technique for sulphomucins mucin and possibly consisted of protein. The results suggest that one type of cell may be able to produce a range of secretory products and to package them variously into secretory granules.     

Localization of cAMP-dependent protein kinase subunits along the secretory pathway in pancreatic and parotid acinar cells and accumulation of the catalytic subunit in parotid secretory granules following beta-adrenergic stimulation

Catalytic (C) and regulatory (RI and RII) subunits of cAMP-dependent protein kinases were localized by immunoelectron microscopy in cisternae of the rough endoplasmic reticulum (rER) and in the Golgi complex of rat pancreas or parotid cells. Zymogen granules of the exocrine pancreas showed C- and RI-immunoreactivity, secretory granules of parotid acinar cells only RII-immunoreactivity. Injection of rats with isoproterenol (IPR) increased in the parotid gland the number of acinar cells with RII-labeled granules. In addition, it led to the appearance of C-immunoreactivity in the condensing vacuoles and secretory granules with a maximum at 24 h after stimulation. This was confirmed by enzyme-linked immunosorbent assay (ELISA) determinations of C- and RII-subunits in secretory granules isolated from stimulated and control parotid glands. The amount of immunoreactive C-subunits in the secretory granules increased further following repeated injections of the beta-agonist. These findings suggest the existence of secretory forms of cAMP-dependent protein kinase R- and C-subunits and their separate regulation.     

Quantitative immunocytochemistry of rat submandibular secretory proteins during chronic isoproterenol administration and recovery

We utilized quantitative electron microscopic immunogold labeling procedures to follow changes in the intragranular content of five secretory proteins of the rat submandibular gland (SMG) during and after chronic treatment with the beta-adrenergic agonist isoproterenol (IPR). Labeling intensities (gold particles/microns2) of acinar cell secretory granules for mucin and glutamine/glutamic acid-rich proteins, major secretory proteins of the normal SMG, showed opposite responses to IPR. Labeling intensities increased for mucin and decreased for glutamine/glutamic acid-rich proteins immediately after IPR injections began, then rapidly returned to control levels after cessation of IPR treatment. SMG Protein C immunoreactivity, found in both acinar and intercalated duct granules, was less affected by IPR. However, opposite changes in labeling intensity were observed between acinar and intercalated duct granules. Labeling intensities for proline-rich proteins, IPR-inducible secretory proteins, increased only after 10 days of stimulation and maintained a high level even after cessation of drug treatment. Type 2 cystatin, another IPR-inducible protein, increased gradually with chronic IPR treatment and decreased slowly during the recovery phase. These results suggest that chronic beta-adrenergic stimulation affects the expression of genes for several rat SMG secretory proteins in a different manner.     

Localization of mitochondrial 60-kD heat shock chaperonin protein (Hsp60) in pituitary growth hormone secretory granules and pancreatic zymogen granules.

We used quantitative immunogold electron microscopy and biochemical analysis to evaluate the subcellular distribution of Hsp60 in rat tissues. Western blot analysis, employing both monoclonal and polyclonal antibodies raised against mammalian Hsp60, shows that only a single 60-kD protein is reactive with the antibodies in brain, heart, kidney, liver, pancreas, pituitary, spleen, skeletal muscle, and adrenal gland. Immunogold labeling of tissues embedded in the acrylic resin LR Gold shows strong labeling of mitochondria in all tissues. However, in the anterior pitutary and in pancreatic acinar cells, Hsp60 also localizes in secretory granules. The labeled granules in the pituitary and pancreas were determined to be growth hormone granules and zymogen granules, respectively, using antibodies to growth hormone and carboxypeptidase A. Immunogold labeling of Hsp60 in all compartments was prevented by preadsorption of the antibodies with recombinant Hsp60. Biochemically purified zymogen granules free of mitochondrial contamination are shown by Western blot analysis to contain Hsp60, confirming the morphological localization results in pancreatic acinar cells. In kidney distal tubule cells, low Hsp60 reactivity is associated with infoldings of the basal plasma membrane. In comparison, the plasma membrane in kidney proximal tubule cells and in other tissues examined showed only background labeling. These findings raise interesting questions concerning translocation mechanisms and the cellular roles of Hsp60.     

Localization of cellular regulatory proteins using postembedding immunogold labeling

Cyclic AMP-dependent protein kinase (cAPK) mediates the effects of catecholamines and hormones that cause elevation of intracellular cyclic AMP levels. The holoenzyme is a tetramer consisting of catalytic (C) and cyclic AMP-binding regulatory (R) subunits. The type I and type II cAPK isoenzymes are defined by R subunits (RI and RII) of differing molecular weight, primary structure, and cyclic AMP-binding properties. Postembedding immunogold labeling procedures and specific polyclonal and monoclonal antibodies to RI, RII, and C were used to study the subcellular distribution of cAPK subunits in several tissues. In the rat parotid gland, both RI and RII were present in the cytoplasm, nuclei, and secretory granules of the acinar cells, whereas secretory granules of intercalated and striated duct cells were poorly labeled. These results confirmed that the acinar secretory granules are the source of R subunits previously identified in saliva by specific photoaffinity labeling techniques. Zymogen granules of pancreatic acinar cells and secretory granules of seminal vesicle cells were labeled with antibody to RII. Pancreatic and seminal fluids were shown to contain cyclic AMP-binding proteins. The granules of several endocrine cells (pituitary, pancreatic islet, intestinal) also labeled with RII antibody. Double labeling of ovarian granulosa cells showed that both RI and C were present in the nuclei and cytoplasm. The localization of cAPK subunits revealed by postembedding immunogold labeling is consistent with the postulated regulatory functions of these proteins in gene expression, cell proliferation, exocytosis, and various metabolic events The widespread occurrence of cAPK subunits in secretory granules and their release to the extracellular environment suggests that they play an important role in secretory cell function.     

Absence of trypsinogen autoactivation and immunolocalization of pancreatic secretory trypsin inhibitor in acinar cells in vitro

Summary To establish the significance of the addition of trypsin inhibitors to pancreatic acinar cells maintained in vitro, cells were cultured in the presence or absence of soybean trypsin inhibitor. Both cultures exhibited similar growth pattern, ultrastructural appearance, as well as secretory properties. Moreover, there was no evidence of trypsinogen activation in the culture medium. Using the immunocytochemical approach, pancreatic secretory trypsin inhibitor antigenic sites were revealed with specific polyclonal and monoclonal antibodies. The results obtained demonstrated that this trypsin inhibitor is in fact a typical pancreatic secretory protein being processed through the endoplasmic reticulum-Golgi-granule secretory pathway of the acinar cells in rat and human tissues. While the polyclonal antibody yield labelings of increasing intensities along the secretory pathway, the monoclonal one probably due to the molecular nature of its specific antigenic determinant, gave higher labelings in the endoplasmic reticulum. In conclusion the present study has shown that pancreatic acinar cells secrete a specific pancreatic trypsin inhibitor which most probably is involved in the mechanism to prevent trypsinogen activation.     

Differential expression of proline-rich proteins in rabbit salivary glands.

Salivary glands synthesize and secrete an unusual family of proline-rich proteins (PRPs) that can be broadly divided into acidic and basic PRPs. We studied the tissue-specific expression of these proteins in rabbits, using antibodies to rabbit acidic and basic PRPs as well as antibodies and cDNA probes to human PRPs. By immunoblotting, in vitro translation, and Northern blotting, basic PRPs could be readily detected in the parotid gland but were absent in other salivary glands. In contrast, synthesis in vitro of acidic PRPs was detected in parotid, sublingual, and submandibular glands. Ultrastructural localization with immunogold showed heavy labeling with antibodies to acidic PRPs of secretory granules of parotid acinar cells and sublingual serous demilune cells. Less intense labeling occurred in the seromucous acinar cells of the submandibular gland. With antibodies to basic PRPs, the labeling of the parotid gland was similar to that observed with antibodies to acidic PRPs, but there was only weak labeling of granules of a few sublingual demilune cells, and no labeling of the submandibular gland. These results demonstrate a variable pattern of distribution of acidic and basic PRPs in rabbit salivary glands. These animals are therefore well suited for study of differential tissue expression of PRPs.     

Localization of mitochondrial DNA encoded cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase subunits I and II in rat pancreatic zymogen granules and pituitary growth hormone granules

Cytochrome c oxidase (COX) complex is an integral part of the electron transport chain. Three subunits of this complex (COX I, COX II and COX III) are encoded by mitochondrial (mit-) DNA. High-resolution immunogold electron microscopy has been used to study the subcellular localization of COX I and COX II in rat tissue sections, embedded in LR Gold resin, using monoclonal antibodies for these proteins. Immunofluorescence labeling of BS-C-1 monkey kidney cells with these antibodies showed characteristic mitochondrial labeling. In immunogold labeling studies, the COX I and COX II antibodies showed strong and specific mitochondrial labeling in the liver, kidney, heart and pancreas. However, in rat pancreatic acinar tissue, in addition to mitochondrial labeling, strong and specific labeling was also observed in the zymogen granules (ZGs). In the anterior pituitary, strong labeling with these antibodies was seen in the growth hormone secretory granules. In contrast to these compartments, the COX I or COX II antibodies showed only minimal labeling (five- to tenfold lower) of the cytoplasm, endoplasmic reticulum and the nucleus. Strong labeling with the COX I or COX II antibodies was also observed in highly purified ZGs from bovine pancreas. The observed labeling, in all cases, was completely abolished upon omission of the primary antibodies. These results provide evidence that, similar to a number of other recently studied mit-proteins, COX I and COX II are also present outside the mitochondria. The presence of mit-DNA encoded COX I and COX II in extramitochondrial compartments, provides strong evidence that proteins can exit, or are exported, from the mitochondria. Although the mechanisms responsible for protein exit/export remain to be elucidated, these results raise fundamental questions concerning the roles of mitochondria and mitochondrial proteins in diverse cellular processes in different compartments.     

The fine structural localization of peroxidase activity in digestive organs of rats and mice

Summary An endogenous peroxidase activity is demonstrated in acinar cells of the salivary gland and epithelial cells of the colonic crypt of normal rats and mice using electron microscopic histochemistry. The main site of the enzymatic activity is cisternae of the rough endoplasmic reticulum including those of the nuclear envelope, while the intensity of the activity is greatly variable among cell types. Some vesicular and cisternal elements of the Golgi apparatus and secretory granules exhibit the reaction, but it is not consistent in all cells with the peroxidase-positive endoplasmic reticulum. It is very interesting that the peroxidase activity is positive in the rough endoplasmic reticulum-Golgi complex-secretory granule system (EGG system) of the cells located at the beginning and the end of the digestive tract. This suggests a peroxidase-dependent anti-infectious mechanism.Some large and small membrane-limited non-secretory granules and mitochondria also reacted.     

Immunocytochemical localization of nerve growth factor (NGF) in the submandibular gland of adult mice by light and electron microscopy

Summary Nerve growth factor (NGF) was localized in the submandibular gland of adult male mice by a direct immunocytochemical method using highly purified antibodies against NGF coupled to horseradish peroxidase. In light microscopic sections the reaction product was entirely confined to the cells of the secretory tubules. The acinar part of the gland was free of reaction product. This finding was confirmed by electron microscopy. Within the cells NGF was localized exclusively in the apical secretory granules.No reaction was observed in the rough endoplasmic reticulum, the Golgi region or in the granules of the basal part of the cells. This observation favours the assumption that NGF is derived from a precursor molecule and that the precursor is transformed into immunologically active NGF within the secretory granules during their transport from the basal to the apical part of the tubular cells. Stimulation of the submandibular gland with carbachol (2 mg/kg) led to a massive release of the content of the secretory granules, including NGF, into the salivary duct.We wish to thank Dr. C. Rufener, Geneva, for communicating to us a new method of antibodyperoxidase coupling and Mrs. M. Durand-Wenger for her excellent technical assistance. This study was supported by the Swiss National Foundation for Scientific Research (Grant Nr. 3.432.74)     

Characterization of immortalized rabbit lacrimal gland epithelial cells

Summary To establish an immortalized lacrimal gland epithelial cell line, the orbital lacrimal glands of normal New Zealand White rabbits were multiply injected with an immortalizing amphotropic retroviral vector (LXSN16E6E7) containing the E6 and E7 genes of human papillomavirus type 16. Lacrimal glands were removed after 2 d and acinar epithelial cells were isolated and cultured on Matrigel-coated 60 mm² plates containing DMEM-F12 supplemented with 5% Nu-serum V. Transformed cells were selected in G418 sulfate for 7 d and passaged. Morphology of the immortalized cells was similar to that described for normal acinar cells both in vivo and in vitro, with rough endoplasmic reticulum and secretory granules. These characteristics remained unchanged and the cells continued to exhibit typical polygonal epithelioid structure. The cells have been maintained in culture for 14 mo. and have gone through 58 passages without loss of proliferation or epithelial cell characteristics. Immunohistochemistry and Western blots showed positive reactivity to secretory component, transferrin, and transferrin receptor, which are typical proteins found in the lacrimal gland. Functional analysis by stimulation with a cholinergic agonist, carbachol (100 μM), resulted in a significant release of protein. This is the first report of an immortalized rabbit lacrimal epithelial cell. These cells will provide a valuable tool for the molecular analysis of lacrimal gland epithelial cell functions.     

Cytochemical localization of terminal N-acetyl-D-galactosamine residues in cellular compartments of intestinal goblet cells: implications for the topology of O-glycosylation

The O-linked oligosaccharides of mucin-type glycoproteins contain N- acetyl-D-galactosamine (GalNAc) that is not found in N-linked glycoproteins. Because Helix pomatia lectin interacts with terminal GalNAc, we used this lectin, bound to particles of colloidal gold, to localize such sugar residues in subcellular compartments of intestinal goblet cells. When thin sections of low temperature Lowicryl K4M embedded duodenum or colon were incubated with Helix pomatia lectin- gold complexes, no labeling could be detected over the cisternal space of the nuclear envelope and the rough endoplasmic reticulum. A uniform labeling was observed over the first and several subsequent cis Golgi cisternae and over the last (duodenal goblet cells) or the two last (colonic goblet cells) trans Golgi cisternae as well as forming and mature mucin droplets. However, essentially no labeling was detected over several cisternae in the central (medial) region of the Golgi apparatus. The results strongly suggest that core O-glycosylation takes place in cis Golgi cisternae but not in the rough endoplasmic reticulum. The heterogenous labeling for GalNAc residues in the Golgi apparatus is taken as evidence that termination of certain O- oligosaccharide chains by GalNAc occurs in trans Golgi cisternae.     

Simultaneous apocrine and merocrine secretion in the rat coagulating gland

The coagulating gland of the rat synthesizes two prevalent secretory proteins (transglutaminase and 115 K) that are discharched in a different manner, one being secreted in an apocrine fashion (transglutaminase) and the other one in a merocrine way (115 K). Differences in the intra- cellular pathway and the release of either protein were studied using immunofluorescence on semithin sections, immunoelectron microscopy of preembedding-processed chopper sections and postembedding-processed ultrathin sections of rat coagulating gland. Immunohistochemical staining using an anti-transglutaminase antibody resulted in dense labeling of the cytoplasm of secretory cells and their apical blebs, whereas the cisternae of the rough endoplasmic reticulum and the Golgi apparatus were completely unlabeled. When, on the contrary, the anti-115 K antiserum was used, dense labeling of the cisternae of the rough endoplasmic reticulum, the Golgi apparatus, and the secretory granules was seen. Intraluminal secretion was also labeled, but the secretory blebs remained unlabeled. Our findings show that, in the coagulating gland of the male rat, the two secretory proteins studied are processed in parallel, but at completely different intracellular pathways. They are released via different extrusion mechanisms. Transglutaminase is synthesized outside the endoplasmic reticulum, reaches the apical cell pole by free flow in the cytoplasm, and is released via apocrine blebs, the membranes of which appear to be derived from the apical plasma membrane. The protein 115 K, on the other hand, follows the classic route, being synthesized within the cisternae of rough endoplasmic reticulum, subsequently glycosylated in the Golgi apparatus, and released in a merocrine fashion. The mutual exclusion of the two secretory pathways and the regulation of the alternative release mechanism are still unresolved issues.     

Subcompartment sugar residues of gastric surface mucous cells studied with labeled lectins.

We examined the intracellular localization of sugar residues of the rat gastric surface mucous cells in relation to the functional polarity of the cell organellae using preembedding method with several lectins. In the surface mucous cells, the nuclear envelope and rough endoplasmic reticulum (rER) and cis cisternae of the Golgi stacks were intensely stained with Maclura pomifera (MPA), which is specific to alpha-Gal and GalNAc residues. In the Golgi apparatus, one or two cis side cisternae were stained with MPA and Dolichos biflorus (DBA) which is specific to terminal alpha-N-acetylgalactosamine residues, while the intermediate lamellae were intensely labeled with Arachis hypogaea (PNA) which is specific to Gal beta 1,3 GalNAc. Cisternae of the trans Golgi region were also stained with MPA, Ricinus communis I (RCA I) which is specific to beta-Gal and Limax flavus (LFA) which is specific to alpha-NeuAc. Immature mucous granules which are contiguous with the trans Golgi lamellae were weakly stained with RCA I, while LFA stained both immature and mature granules. The differences between each lectin's reactivity in the rough endoplasmic reticulum, in each compartment of the Golgi lamellae and in the secretory granules suggest that there are compositional and structural differences between the glycoconjugates in the respective cell organellae, reflecting the various processes of glycosylation in the gastric surface mucous cells.     

The effect of aging on the rat submandibular gland: an ultrastructural, cytochemical and biochemical study

The effect of aging on the rat submandibular gland was studied by using ultrastructural, ultrastructural cytochemical and biochemical techniques. There was an age-related clumping of the nucleolar-associated and peripheral chromatin in many of the acinar cells and a decrease in the number of cisternae of rough endoplasmic reticulum. Many aged acinar cells were binucleated. There was also an age-related increase in pigment granules throughout the gland. These membrane bound granules consisted of a lipid droplet and an associated dense cap which had a granular matrix and pigment droplets. The lead capture method for acid phosphatase activity demonstrated that activity was associated with the granular matrix of the dense cap. These results were correlated with the age-related increase in acid phosphatase activity as determined by colorimetric procedures. There was an age-related increase in the number of cells characterized by small secretory granules. These cells were found as part of the intercalated ducts or at the junction of the duct with the acini. Oncocytes were also found as part of the parenchyma of the aged submandibular gland. These cells were characterized by the pleomorphic mitochondria that fill their cytoplasm. Occasionally, cells that possessed extraordinary numbers of mitochondria and small secretory granules were also observed. The determinations of total DNA and RNA revealed and age-related decrease in RNA while there was no significant change in the concentration of DNA.     

The epidermis of Timarete filigera (Polychaeta, Cirratulidae): histochemical and ultrastructural analysis of the gland cells

The aim of this study was to verify whether different living conditions of Polychaeta are correlated with morphological and functional differences in the organization of the integument. For this purpose, we decided to study the epidermis of Timarete filigera, a non-tubicolous polychaete. With this objective in mind, we have identified the various cellular types responsible for mucous secretion in the epidermis of this species and defined the histochemical composition of the mucus produced by different types of gland cells. Three types of gland cells have been identified by histochemical and ultrastructural studies in the epidermis of this polychaete. The histochemistry was carried out using standard techniques and peroxidase-labelled lectins. In type 1 cells, the secretory granules contain neutral glycoproteins with glucosidic residues of GalNAc, Galbeta 1,3 GalNAc, glucosidic and/or mannosidic residues. In type 2 cells, the secretory granules contain acid glycoproteins mainly sulphated with glucosidic residues of GalNAc, Galbeta 1,3 GalNAc, glucosidic and/or mannosidic residues, and some terminal sialic acid. In type 3 cells, the residual granules have the same chemical composition as that of granules present in type 2 cells. The secretion of these glandular mucous cells consists of mainly sulphated acidic glycoproteins and GAG resistant to testis jaluronidase. In these cells, the residual granules have the same chemical composition as that of their secretion. The heterogeneity of mucus composition may be correlated with its different functions.