Mucus-glycoproteins (mucins) of the cat trachea: characterisation and control of secretion

Glycoproteins produced by the tracheae of anaesthetized cats were radiolabelled biosynthetically by a pulse administration of Na2 35SO4 and [3H]glucose into the tracheal lumen. Subsequently, radiolabelled secretions were washed from the tracheal lumen. Repeated doses of pilocarpine and then ammonia vapour were given to stimulate secretion. Pilocarpine-stimulated glycoproteins, which came mainly from the submucosal glands, were particularly enriched with 35S. Ammonia-stimulated secretions, which probably came mostly from the microvillous border of the surface epithelium, contained mainly 3H radioactivity but little 35S. Two negatively-charged glycoproteins of different molecular size were identified in the secretions: the larger component was excluded on Sepharose CL-4B and it had a higher 3H 35S ratio than the smaller component which was retarded on Sepharose CL-4B. The relative amount of the smaller component decreased progressively with repeated pilocarpine stimulation and it was not detected in secretions induced by ammonia. Pilocarpine stimulation caused little alteration in carbohydrate composition of the secreted glycoproteins. In response to ammonia, glycoproteins were secreted with a high sialic acid content but quantitatively they represented a small amount of material compared with that induced by pilocarpine. These findings suggest that tracheal glycoproteins from different epithelial-cell sources have distinctive chemical compositions and that their secretions may be independently regulated. The 35S-rich high-molecular-weight glycoproteins from the submucosal glands were of the mucin-type but those derived from the microvillus border may represent a different class of airway glycoproteins from typical epithelial mucins.     

Pulmonary C-fibers reflexly increase secretion by tracheal submucosal glands in dogs

Stimulation of bronchial C-fibers evokes a reflex increase in secretion by tracheal submucosal glands, but the influence of pulmonary C-fibers on tracheal gland secretion is uncertain. In anesthetized dogs with open chests, we sprayed powdered tantalum on the exposed mucosa of a segment of the upper trachea to measure the rate of secretion by submucosal glands. Secretions from the gland ducts caused elevations (hillocks) in the tantalum layer. We counted hillocks at 10-s intervals for 60 s before and 60 s after we injected capsaicin (10-20 micrograms/kg) into the right atrium to stimulate pulmonary C-fiber endings. Right atrial injection of capsaicin increased the rate of hillock formation fourfold, but left atrial injection had no significant effect. The response was abolished by cutting the vagus nerves or cooling them to 0 degree C. We conclude that the reflex increase in tracheal submucosal gland secretion evoked by right atrial injection of capsaicin was initiated as capsaicin passed through the pulmonary vascular bed, and hence that pulmonary C-fibers, like bronchial C-fibers, reflexly increase airway secretion.     

The effect of pH on Alcian Blue staining of epithelial acid glycoproteins. I. Sialomucins and sulphomucins (singly or in simple combinations)

Synopsis This study is concerned with the staining of epithelial acid glycoproteins by Alcian Blue at various pH levels. A detailed analysis of the effect of pH on Alcian Blue staining of epithelial tissues at selected sites was made. Alcian Blue was combined with the periodic acid-Schiff technique, the Alcian Blue being used at pH levels between 2.6 and 0.5.Animal salivary glands, human foetal tracheal gland and epithelial goblet cells of the adult bronchial mucosa were chosen for study because the nature of their acid glycoprotein is known and is relatively simple.In sites containing sialomucin alone, no Alcian Blue staining took place below pH 1.5. A difference was demonstrated between sialidase-sensitive sialomucin which stained only between pH 2.6 and 1.7, and sialidase-resistant sialomucin which stained between pH 2.6 and 1.5. Two types of sulphomucin were identified: the usual one stained with Alcian Blue at all the pH levels studied, and the other, in the canine gland, stained only at the most acid pH levels, that is, between pH 1.5 and 0.5.     

Comparative developmental analysis of the parotid, submandibular and sublingual glands in the neonatal rat.

Analysis of the soluble protein fractions from the rat parotid, submandibular and sublingual glands by polyacrylamide-gel electrophoresis reveals similarities in overall patterns of protein synthesis at birth. Tissue-specific changes in protein and glycoprotein synthesis occur shortly after birth and again at the time of weaning, 21--28 days later. Incorporation of [3H]thymidine into DNA was at its highest after birth and gradually decreased in both the parotid and submandibular gland, whereas [3H]thymidine incorporation in the sublingual gland was low throughout the time of neonatal development. [14C]Leucine incorporation into total protein increased in all glands with age after birth, showing an accelerated rate 21--28 days later. Trichloroacetic acid/phosphotungstic acid-precipitable [3H]fucose in glycoproteins declined over the time of neonatal development in the parotid and submandibular gland, but its incorporation remained higher in the sublingual gland. alpha-Amylase (EC 3.2.1.1) in the salivary glands increased at the time of weaning, as judged by detectability in sodium dodecyl sulphate/polyacrylamide gels and by immune precipitation. Two membrane-bound enzymes, UDP-galactose:2-acetamido-2-deoxy-D-glucosamine 4 beta-galactosyltransferase (EC 2.4.1.22) and UDP-galactose:2-acetamido-2-deoxy-D-galactosaminyl-protein 3 beta-galactosyltransferase (no EC number), undergo tissue-specific change rather than changes induced by physiological stimulation of the salivary glands.     

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.     

Effects of prolactin, progesterone, and 17beta-hydroxy-5alpha-androstan-3-one on squalene production by the preputial gland of the immature female rat

To examine further the previously demonstrated synergism between prolactin and progesterone on preputial glands of hypophysectomized, ovariectomized, immature rats, their effects on squalene production were determined and compared with the ability of 17beta-hydroxy-5alpha-androstan-3-one (DHT) and prolactin to increase the amount of squalene in the preputial glands. Glands from progesterone-treated rats incubated in vitro with [14C]mevalonic acid incorporated radioactivity into squalene (identified by chromatographic mobility) more rapidly than glands from controls or prolactin-treated rats. Using the same in vitro system, glands from prolactin-treated rats incorporated more [14C]acetate into squalene than those from progesterone-treated animals. In addition, results showed that prolactin and DHT increased nonradioactive squalene (identified by mass spectral analysis) content in the gland while progesterone had no effect. It is proposed that prolactin increases preputial gland squalene content by enhancing synthesis of mevalonic acid, while progesterone increases incorporation of mevalonic acid into squalene.     

Histological and histochemical observations on the foot glands in some byssus-bearing bivalves of the Waltair coast

Summary The glands responsible for the formation of the byssus threads inArca symmetrica, Barbatia obliquata andSeptifer bilocularis are the white gland, phenol gland and enzyme gland. Besides these, mucous glands are also present in the sub-epithelia. The size and shape of the cells of these glands vary in one and the same species. From histochemical investigations it has been revealed that these glands contain 1,2-glycol groups in addition to disulphides and sulphhydryls. The white gland secretes a protein material and the phenol gland is rich in phenols. These two combine to form a phenolic protein which is acted upon by a polyphenol oxidase secreted by the enzyme gland and leads to the formation of a byssus thread. The mucous gland cells secrete acid mucopolysaccharides, neutral mucins and glycoproteins.     

Composition of glycoproteins secreted by tracheal explants from various animal species.

1. The acidic and neutral glycoproteins secreted by cultured tracheal explants from pigs, sheep, rats, mice, monkeys, guinea pigs, dogs and chickens were purified and fractionated by column chromatography on DEAE-cellulose and by electrophoresis on cellulose acetate. 2. The ratios of acidic to neutral mucus glycoproteins were compared for the above animals with that of mucus glycoproteins secreted by cultured human bronchi. 3. The observed ratios of acidic to neutral glycoproteins ranged from 4.0 (mouse) to 7.2 (chicken and pig) from cultured tracheae; secreted human bronchial mucus had a ratio of 2.7. 4. The ratio of acidic to neutral glycoproteins secreted by tracheal explants varied with duration of incubation of the trachea in culture.     

Studies on the Indian sand lobster Thenus orientalis (Lund): mucopolysaccharides of the tegumental glands.

The tegumental glands are imbedded in the connective tissue below the epithelium of oesophagus. Each gland is made up of cells which are conical in shape with their narrow ends directed towards the lumen of the gland. In the centre of each gland there is a cavity which communicates with intracellular duct. Similar glands have been found in the hind-gut region also. These glands secrete both acid and neutral mucopolysaccharides and to some extent glycoproteins. The glands are charged mostly with the task of secreting weakly acidic mucopolysaccharides and neutral mucopolysaccharides which are confined to the apices and central cavity of the gland. The acidic nature is partly due to sialic acid and partly due to hyaluronic acid. These weak acids do not seem to play any role in digestion but lubricate the lumen of the oesophagus for easy passage of food and keep the lining of the oesophagus slimy. In the hind-gut they help in binding the faecal matter into pellets.     

Ultrastructural localization of acidic glycoconjugates with the low iron diamine method

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.     

Sulfated mucopolysaccharide synthesis during the development of Rana pipiens

Sulfated mucopolysaccharide (MPS) synthesis during the development of Rana pipiens was studied autoradiographically and biochemically following injection of embryos with ³⁵S-sulfate. ³⁵S-sulfate incorporation can be detected in unfertilized and fertilized eggs. The sulfate-incorporating material accumulates along the periphery of yolk platelets of eggs. During cleavage, the ³⁵S-sulfate-incorporating material accumulates on cell surfaces as well as along the periphery of yolk platelets. Biochemical analysis utilizing the enzymes chondroitinase ABC and AC and nitrous acid degradation indicates that the MPS synthesized during cleavage is approximately 82% heparin and/or heparan sulfate and 18% chondroitin 4-sulfate. During gastrulation, a greatly enhanced incorporation of ³⁵S-sulfate is observed in the invaginating chordamesoderm and lateroventral mesoderm, and by the end of gastrulation enhanced incorporation can be detected in neural tissue. During this period, chondroitin 6-sulfate synthesis is initiated. Incorporation of ³⁵S-sulfate is observed in all tissues of the embryo from the beginning of neurulation through hatching. This ubiquitous incorporation is accompanied by an increase in the relative amount of chondroitin 6-sulfate synthesized. During the period following hatching, incorporation is suppressed in some tissues and enhanced in others so that by the late feeding tadpole stage a very high incorporation is observed only in cartilaginous tissue. These results indicate that sulfated MPS synthesis occurs in all stages of development of Rana pipiens, but that significant changes in the rate of synthesis occur in various cell types during gastrulation and after hatching. The ubiquity of sulfated MPS synthesis during the critical early stages of development and the changes in the pattern of synthesis in various cell types suggest that these molecules are involved in a number of embryonic processes.     

Incorporation of 35S-sulfate and 3H-glucosamine into heparan and chondroitin sulfates during the cell cycle of B16-F10 cells

Changes in glycosaminoglycan composition occurring during the cell cycle were determined in B16-F10 cells sorted flow cytometrically with respect to DNA content. Incorporation of 35S-sulfate into heparan sulfate and chondroitin sulfate of unsorted and G1,S, and G2 +M sorted cells was determined following chondroitinase ABC or nitrous acid treatment; the incorporation into surface material was measured as the difference between the radioactivity of control and trypsin-treated cells. Incorporation of 35S-sulfate and 3H-glucosamine into cetyl pyridinium chloride (CPC)-precipitable material was characterized before and after chondroitinase or nitrous acid treatment by Sephadex G50 chromatography. Long-term (48 h) and short-term (1 h) labeling studies demonstrate that (a) the amount of total cellular chondroitin sulfate is greater than that of heparan sulfate, with larger amounts of unsulfated heparan than chondroitin being present; (b) the rate of turnover of heparan sulfate is greater than that of chondroitin sulfate; (c) greatest short-term incorporation of 3H-glucosamine into CPC-precipitable material occurs during S phase; and (d) the rate of turnover of both heparan sulfate and chondroitin sulfate is decreased in S phase relative to G1 and G2 + M.     

The development of the pouch-like structures at the angles of the mouth in the ontogeny of the hamster Phodopus campbelli]

The development of mouth angle sacciform structure was studied in the ontogenesis of hamster Phodopus campbelli. It was found that the true sacciform structure with all components characteristic for adult animals was formed by the 20th day of postnatal development. Histological data have shown than the sacciform structure is formed in ontogenesis as a result of a complex epidermal transformation involving muscular and connective tissues which comprise the external coat. The structure studied is not formed on the basis of sebaceous glands and cannot be assigned to the class of gland as considered before.     

Studies on the exocrine secretions

Summary The present work attempts to throw further light on the field of exocrine secretion in general and on the salivary gland function in particular. Special attention has been paid to the synthetic products of the mucous-secreting and protein-producing cells making up the submaxillary and the parotid glands. To this end, several exotic animals have been utilized for these experiments, among which tapirs, giant and small ant eaters, two-toed sloths, skunks, racoons, neonatal and adult opossums, armadillos, flying squirrels and South American porcupines. The data reported in this paper concerns the tapirs, ant eaters, opossums, two-toed sloths, skunks and racoons. The histochemical experiments carried out endeavored to identify the chemical products elaborated by the salivary gland cells. In general terms it was found that serous-producing structures synthesize, along with the enzymatic proteins, different amounts of glycoprotein material. These carbohydrate-protein molecules are both of the neutral and acidic type. The parotid glands of ant bears, sloths and skunks secreted an acid mucin identified as a sialoglycoprotein; the parotids of the opossums, racoons and tapirs did not elaborate histochemically detectable acidic polyanions.All the parotid glands produced high amounts of neutral glycoproteins and the zymogen granules were strongly stained by PAS. The parotid glycoproteins were very resistant to enzymatic degradation as noticed after proteolytic digestion with papain and pepsin.With the exception of the basophilia in the parotid of the ant bears (only slightly affected) neuraminidase did not modify the PAS and AB stainings in any of the serous-producing structures. Conversely, the mucin of the submaxillary glands was quite susceptible to proteolysis and the histochemical approach indicated that it was composed primarily of a sialoglyco-protein. The only exception was represented by the opossum submaxillary whose mucin showed strong chemical affinity with the parotid secretion. The basophilic staining of the submaxillary glands in general was very susceptible to neuraminidase. Of interest was the fact that the PAS reaction was also drastically abolished in several animal glands. This data further confirmed that 1) the glycoprotein present in these glands was primarily represented by sialic acid and indicated that 2) this acid is, per se, PAS-positive.The excretory system in some animal glands was highly secreting. The type of material elaborated by the cells making up the ducts was almost exclusively represented by neutral glycoproteins indicating, thus, that the type of secretion produced by the ductal cells was chemically similar to that of the parotid.These results indicate clearly that the old classification which considers these glands serous or mucous is erroneous. There is no purely protein-secreting gland since all, even though in different measure, elaborate and synthesize glycoprotein material.Supported by Grant No. DE-02110-04-05 of the National Institutes of Public Health, Bethesda, Md.     

Polyomavirus major capsid protein VP1 is modified by tyrosine sulfuration.

Polyomavirus was propagated in primary mouse kidney cell monolayers and 35S-sulfate labeled by maintaining the infected cells in serum-free Eagle medium supplemented with 35S-labeled sodium sulfate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CsCI gradient-purified 35S-sulfate-labeled virions followed by fluorography indicated that the polyomavirus-coded major capsid protein VP1 incorporated this radiolabel. Two-dimensional gel electrophoresis followed by fluorography revealed 35S-sulfate incorporation into only two of the six VP1 isoelectric species (E and F). Amino acid analysis of 35S-sulfate labeled VP1 by enzymatic hydrolysis followed by two-dimensional thin-layer electrophoresis revealed the presence of 35S-sulfate-labeled tyrosine-O-sulfate.     

Fine structure and function of the prosomal glands of the two-spotted spider mite,Tetranychus urticae (Acari,Tetranychidae)

Summary The prosomal glands of Tetranychus urticae (Acari, Tetranychidae) were examined light and electron microscopically. Five paired and one unpaired gland are found both in females and males. The silk spinning apparatus consists of paired silk glands which extend laterally on both sides of the esophagus into the pedipalps. There, they enter the terminal silk gland bag which opens into a silk bristle at the apex of the pedipalps. The salivary secretions are formed in three paired glands which have an interconnecting duct, the podocephalic canal. The dorsal podocephalic glands may produce a serous secretion, the anterior podocephalic glands a mucous secretion, and the coxal organ may add a liquid, ion-rich secretion. These secretions pass the podocephalic canal and reach the mouth at the apex of the gnathosome. The function of the paired tracheal organs and the unpaired tracheal gland is still unclear. The tracheal gland may produce a secretion which facilitates the movement of the fused chelicerae and the stylets.This study was financed by a grant from the Deutsche Forschungsgemeinschaft (DFG Se 162/12)     

Interaction of the tracheal tubules of Scutigera coleoptrata (Chilopoda,Notostigmophora) with glandular structures of the pericardial septum

Notostigmophora (Scutigeromorpha) exhibit a special tracheal system compared to other Chilopoda. The unpaired spiracles are localized medially on the long tergites and open into a wide atrium from which hundreds of tracheal tubules originate and extend into the pericardial sinus. Previous investigators reported that the tracheal tubules float freely in the hemolymph. However, here we show for the first time that the tracheal tubules are anchored to a part of the pericardial septum. Another novel finding is this part of the pericardial septum is structured as an aggregated gland on the basis of its specialized epithelium being formed by hundreds of oligocellular glands. It remains unclear whether the pericardial septum has a differently structure in areas that lack a connection with tracheal tubules. The tracheal tubules come into direct contact with the canal cells of the glands that presumably secrete mucous substances covering the entire luminal cuticle of the tracheal tubules. Connections between tracheae and glands have not been observed in any other arthropods.     

Osteoblasts during various functional states

At the electron microscopical investigation of osteoblasts in different zones of osteogenesis (enchondral foci, metaphyses, endosteum) in the rat and rabbit femoral bone it has been revealed that their population is heteromorphic. As demonstrate cytochemical data and radioautography, using 3H-uridine, 3H-glycine, 35S-sulfate, 45Ca, results of measurements in osteoblast population, 4 morpho-functional states (or types) are defined. In areas of an intensive osteoplastic process there are young osteoblasts (I type), mature functionally active osteoblasts (II type), osteoblasts with a hypertrophic endoplasmic network (cell-depots of the secrete--III type). In preosteoblasts and osteoblasts of the I type a higher than in osteoblasts of other types intensity of 35S-sulfate incorporation and alkaline phosphatase activity is revealed. In osteoblasts of the II type processes of biosynthesis of collagenous proteins predominate. Osteoblasts of the III type are subjected to destruction during secretion process. In the areas where osteopoesis is dying away, osteoblasts of the I and II types transform into a poorly active state, concerning specific biosynthesis (osteoblasts of the IV type). Presence of osteoblasts having various functional states in the areas of intensive osteopoiesis, depends on certain asynchronity of specific processes of biosyntheses, that occur in them. The morpho-functional states described are regarded as a specific peculiarity in function of collagene-producing cells.     

Fatty acid acylation of salivary mucin in rat submandibular glands

The acylation of salivary mucin with fatty acids and its biosynthesis was investigated by incubating rat submandibular salivary gland cells with [3H]palmitic acid and [3H]proline. The elaborated extracellular and intracellular mucus glycoproteins following delipidation, Bio-Gel P-100 chromatography, and CsCl equilibrium density gradient centrifugation were analyzed for the distribution of the labeled tracers. Both preparations gave single bands at the CsCl density of 1.48, in which carbohydrate peaks coincided with that of the labels. The [3H]palmitic acid in these glycoproteins was susceptible to cleavage by alkali and hydroxylamine, thus indicating the ester nature of the bond. With both intracellular and extracellular glycoproteins deacylation caused the glycoproteins to band in the CsCl gradient at a density of 1.55. The incorporation of both markers into mucus glycoprotein increased steadily with time up to 4 h, at which time about 65% of [3H]palmitate and [3H]proline were found in the extracellular glycoprotein and 35% in the intracellular glycoprotein. The incorporation ratio of proline/palmitate, while showing an increase with incubation time in the extracellular glycoprotein, remained essentially unchanged with time in the intracellular glycoprotein and at 4 h reached respective values of 0.14 and 1.12. The fact that the proline/palmitate incorporation ratio in the intracellular glycoprotein at 1 h of incubation was 22 times higher than in the extracellular and 8 times higher after 4 h suggests that acylation occurs intracellularly and that fatty acids are added after apomucin polypeptide synthesis. As the incorporation of palmitate within the intracellular mucin was greater in the mucus glycoprotein subunit, it would appear that fatty acid acylation of mucin subunits preceeds their assembly into the mucus glycoprotein polymer.     

The effects of testosterone administered neonatally on the incorporation of 3H-leucine into protein by thyroid gland of rat: a neonatal priming.

Rats were castrated on day 2 after birth, given one injection of testosterone pronate (TP: 2.5 mg) or oil just after operation and then received TP or oil when adult. 3H-Leucine was injected intravenously 2.5 hours before killing. The incorporation of labelled amino acid into protein was investigated in the thyroid glands. Males receiving TP both neonatally and when adult incorporate twice as much labelled leucine into protein as compared to their counterparts. Grain density was highest in the thyroid gland of early hormone treated rats when challanged with testosterone in adulthood. Thyroid gland priming by early hormone treatment has been discussed.