414
BIOCHEMICAL SOCIETY TRANSACTIONS
holocrine secretion is not restricted to the well-known sebaceous glands. It is difficult to discern between the content of whole disintegrated cells and the mature product liberated from viable cells by secretion. The extramural glands of the tracts do not raise such difficulties. Although the structural aspects and kinetics of secretion have become rather well known, the underlying mechanism of membrane fusion and its regulation are by no means understood. The key to a better understanding might be found in the study of fusing membranes. The use of cultures of free secretory cells seems to be advantageous. The study of the conditions that favour fusions between cellular membranes, fragmentation vesicles and liposomes might also be expected to throw light on one of the most fundamental processes, controlling so many functions essential for organized life. Bauduin, H., Colin, M. & Dumont, J. E. (1969)Biochim. Biophys. Acta 174,722-733 Bock, E. & Jorgensen, 0. S. (1975)FEBSLett. 52,37-39 Bogart, B. J. (1975)J. Ultrastruct. Res. 52, 139-155 Bolender, R. P. (1974)J. Cell Biol. 61, 269-287 Davis, B. K. (1974)J. Reprod. Fertil. 41,241-244 de Camilli, P., Peluchetti, D. & Meldolesi, J. (1974)Nature (London) 248, 245-247 Geuze, J. J. & Kramer, M. F. (1974)Cell Tissue Res. 156, 1-20 Geuze, J. J., Kramer, M. F. & de Man, J. C. H. (1976) in Mammalian Cell Membranes (Jamieson, G . A. & Robinson, D. M., eds.), vol. 2,chapter 2,Buttenvorth, London Geuze, J. J. & Slot, J. W. (1976)Biol. Reprod. 15, 118-125 Harrop, T. 3. & Garrett, J. R. (1974)Cell Tissue Res. 154, 135-150 Jamieson, J. D.& Palade, G. E. (1971)J. Cell Biol. 48,503-522 Kim, S. K., Nasjleti, L. E. & Han, S. S. (1972)J. Ultrastruct. Res. 38, 371-389 Kramer, M. F. & Poort, C. (1972)J. CeN Biol.52, 147-158 Meldolesi, J. (1974)J. Cell Biol. 61, 1-13 Meldolesi, J. & Cova, D. (1972)J. Cell Biol. 55, 1-18 Meldolesi, J., Jamieson, J. D. & Palade, G. E. (1971)J. Cell Biol. 49, 130-158 Palade, G. E. & Bruns, R. R. (1968)J. Cell Biol. 37, 633-649 Raff, M., unpublished work, cited by Bretscher, M. S. (1976)Nature (London)261, 366-367 Satir, B., Schooley, C. & Satir, P. (1973)J. Cell Biol. 56, 153-176 Wallach, D.,Kirshner, N. & Schramm, M. (1975)Biochim. Biophys. Acta 375, 87-105 White, D. A. & Hawthorne, J. N. (1970)Biochem. J. 120,535-538 Winkler, H., Hortnagl, H., Rufener, C., Nakane, P. K. & Schneider, F. H. (1974) in CytopharmacologyofSecretion (Ceccarelli, B., Meldolesi, J. & Clementi, F., eds.), pp. 127-140, Raven Press, New York
Neurophysiological Control of Glycoprotein Secretion in the Tracheal Epithelium P. W. KENT* and J. G. WIDDICOMBEt *Glycoprotein Research Unit, University of Durham, Durham DH1 3LH, and t Department of Physiology, St. George’s Hospital Medical School, London S W17 OQT,U.K.
The formation of a mucus barrier, external to a mammalian epithelial tissue, is the outcome of two major processes, the biochemical pathways of glycoprotein assembly and the cellular physiology of secretion, i.e. glycoprotein storage and export. Though these processes are subject to their distinctive modes of regulation, stimulation and inhibition, they are inter-related by mechanisms which are as yet not understood. The tracheal epithelium is a promising tissue for the investigation of both processes. Apart from the obvious interest in a connexion with respiratory disorders, the innervation of the trachea is well known in a number of species and is accessible for experimental intervention. The tracheal epithelial mucus in the cat, rabbit, dog 1977
567th MEETING, DURHAM
41 5
Table 1. Carbohydrate composition of principal tracheal glycoproteins (jiractionated on DEAE-cellulose DE-52) Values are expressed as molar ratios relative to N-acetylglucosamine (=l); sialic acid is measured as N-acetylneuraminic acid. Rabbit*
Fucose Galactose N-Acetylglucosamine N-Acet ylgalactosamine Sialic acid Sulphate * From organ culture. t From tracheal perfusion.
Dog*
. (Soluble) (Insoluble) (Soluble) 0.84 0.37 0.23 1.03 0.92 1.60 1 .oo 1 .oo 1.oo 1.83 1 .oo 0.74 0.93 0.77 0.28
++
++
++
Catt
Gooset
(Soluble) (Soluble) 0.65 1.56 1 .oo 1.30 1 .oo 1 .oo 1.25 0.94 0.36 0.40 U
++
Table 2. Ratio of [35S]sulphate-and [3Hlglucose-derived radioactivity in cat tracheal mucins before and after administration of irritants Separate mucin collections were pooled as indicated. Controls (collections 1-5) represent the first five collections of radioactive mucin before addition of pilocarpine. Three fractions, collections (7), (8+9) and (12-1 5), were examined after pilocarpine treatment (collection 6). Control collections (lo), (11) and (16) represent mucin samples taken before and after the second phase of pilocarpine stimulation (collections 12-15). Pooled fraction (collections 17 and 18) was taken after NH, treatment. Irritant None (controls) Pilocarpine Pilocarpine Pilocarpine Pilocarpine None (controls) NH3
Collection (1-5)
(6) (7) (8,9) (12-15) (10, 11, 16) (17918)
Ratio 3H/35S 6.23 2.07 3.19 3.73 4.46 5.03 11.11
and in man is a mixed secretion arising from at least three cell types, namely epithelial goblet cells and mucous and serous cells of the submucosal glands. It has been of interest to explore whether these different cell types synthesize chemically different glycoproteins, and whether each is separately innervated, responding distinctively to irritants and stimulation. A connexion between glycoprotein secretion and nervous stimulation has long been known and there is some evidence of accompanying variations in chemical composition. This was first suggested by Dische et af. (1962) and Dische (1963), who showed that electrical or pilocarpine stimulation of dog submaxillary gland increased the fucose/sialic acid ratio. This has been further investigated (Winzler, 1973 ; Lombart & Winzler, 1975) as discussed by Phelps & Young (1977). The development of a new experimental approach has enabled mucus output to be measured by tracheal lavage of anaesthetized cats to which radioisotopic precursors, e.g. [35S]sulphate or [3H]glucose, had been administered into the blood or into the tracheal lumen (Gallagher et al., 1975). Sympathetic-nerve stimulation and sympatho-
Vol. 5
416
BIOCHEMICAL SOCIETY TRANSACTIONS
Table 3. Comparison of sugar composition of cat miicins obtained after administration of irritants Values were obtained by g.1.c. analysis of samples after ion-exchange fractionation on DE52 DEAE-cellulose. Results are expressed as molar ratios, relative to N-acetylglucosamine (=l). Treatment Mucus collections Sugars Fucose Galactose N-Acetylgalactosamine N-Acetylglucosamine Sialic acid
... ...
Pilocarpine (6)
Control
1.51 1.64 0.70
I .25 I .45 0.80
1 .oo
0.62
(1-5)
1 .oo 0.55
Ammonia (17 and 18) 0.12 1.15 0.65 I .oo 1.15
mimetic amines greatly enhanced output of 35S-labelledmucus above resting values. After either excitation the amount of label in the secretion rapidly returned to that of the resting state. These effects were prevented by 8-adrenergic blocking agents, e.g. propanolol, but not by a-blockade by using phentolamine. Stimulation of the parasympathetic nerve (vagal) also resulted in increased mucus output, of an order similar to that of the sympathetic effects. In this case, the effects were blocked by atropine, whereas pilocarpine, a parasympathomimetic agent, greatly increased the mucus outflow. Fractionation of the mucus glycoproteins on DEAE-cellulose DE52 and on Sephadex G-200 (Gallagher & Kent, 1975) showed two glycoproteins to be present, the electrophoretically less mobile fraction carrying 35S label. The composition of the material from the cat and other species is given in Table 1. Histological examination showed that most mucous and goblet cells produced a [35S]sulphate-labelled glycoprotein. Radioautography showed that pilocarpine and nervous stimulation caused release of labelled glycoprotein from submucosal glands (serous and mucous cells) rather than from epithelial goblet cells. Subsequent administration of NH3 to the same trachea promoted secretion from goblet cells (Gallagher et al., 1977). In a series of double-labelling experiments, Na235S04(1 mci) and ~-[l-~H]glucose (0.25mCi) were introduced, in medium, into the cat tracheal lumen. The KrebsHenseleit (1932) medium which filled the trachea was replaced hourly. The doublelabelled mucus attained a steady rate of formation after lhh. Pilocarpine (6.5pg/ml in medium) was given four times at intervals of 15 min. This was followed by two exposures to NH3 vapour (1 :loo) in air. After exposure to each stimulant the secreted mucus was washed from the trachea and examined chemically. The radioisotopic ratios are shown in Table 2 and the sugar composition of fractions is given in Table 3. It is evident that pilocarpine evokes a more highly sulphated much from submucosal glandular cells. Repeated pilocarpine stimulation results in diminished sulphation without pronounced effect on molecular size. The glycoprotein released from goblet cells by NH3 is distinctive in having a low sulphate content and high sialic acid. The cellular origins of mucus in the goose trachea are quite different from those of mammals in that only goblet cells are present. The ~ U C O L I secretion S here was stimulated by acetylcholine, an effect which was completely blocked by atropine. Neither a- nor &stimulant sympathomimetic amines affected the rate of secretion. Examination of the goose tracheal glycoproteins after fractionation by gel filtration revealed that fucose was absent but that mannose was consistently present (Phipps et al., 1976). The results suggest that in mammals the tracheal mucus is a mixture of glycoproteins of different cellular origins, that nervous control of mucous and serous cells in bronchial glands is exerted through the parasympathetic system, and that the various cellular sites are differently capable of stimulation by chemical irritants. 1977
567th MEETING, DURHAM
417
Dische, Z. (1963) Ann. N. Y. Acad. Sci. 106, 259-270 Dische, Z., Pallavicini, C., Kavaski, H., Smirnow, N., Cizek, L. & Chien, S. (1962) Arch. Biochem. Biophys. 97,459-469
Gallagher, J. T. & Kent, P. W. (1975) Biochem. J. 148, 187-196 Gallagher, J. T., Kent, P. W., Passatore, M., Phipps, R. J. & Richardson, P. S. (1975) Proc. R. Soc. London, Ser. B 192,49-76
Gallagher, J. T., Kent, P. W., Phipps, R. J. & Richardson, P. S. (1977) in Mucus in Healrh and Disease (Parke, D. V. & Elstein, M., eds.), Plenum, London, in the press Krebs, H. A. & Henseleit, K. (1932) Hoppe-Seyler’s 2. Physiol. Chem. 210, 33-66 Lornbart, C . G . & Winzler, R. J. (1975) Eur. J. Biochem. 49, 77-86 Phelps, C. F. & Young, A. M. (1977) Biochem. Soc. Trans. 5, 417-419 Phipps, R. J., Richardson, P. S . , Corfield, A., Gallagher, J. T., Jeffery, P. K., Kent, P. W. & Passatore, M. (1976) Philos. Trans. R. Soc. London, Ser. B in the press Winzler, R. J. (1973) in Membrane Mediated Information (Kent, P. W., ed.), vol. 1, pp. 3-19, Medical and Technical Publishing Co., Lancaster
The Regulation of Mucin Production in the Feline Submaxillary Gland CHARLES F. PHELPS and AILEEN M. YOUNG Department of Biological Sciences, University of Lancaster, Lancaster LA1 4 YQ, U.K.
The submaxillary gland offers a complex system in which to study the control of epithelial mucin secretion. This complexity has a number of roots. Apart from the large number of different animals whose submaxillary glands and their contents have been studied, each gland can exist in a gradation of functioning states between the wholly quiescent and the fully active. This activity may be promoted by sympathetic or parasympathetic innervation as well as by chemical means; the components produced by the gland are many and diverse, and include water, inorganic electrolytes, enzymes and growth-promoting factors as well as complex glycoproteins. Though information exists on many of the components mentioned above, the integrated knowledge of the functioning of the gland is largely fragmentary. For this reason we decided to study a preparation in vivo of cat submaxillary gland. The surgical procedure exposes one gland and enables cannulation of the duct, and also of the blood supply to the gland. Both sympathetic and parasympathetic innervation are exposed, allowing external experimental control to be exercised over the saliva flow. The contralateral gland that was merely exposed acted as control. With this experimental protocol we have a system in which: (a) there is production of a few characterizable glycoproteins; (b) the production is variable in quality and quantity, depending on degree and type of stimulus; ( c ) the preparation is isolatable from other humoral factors such as hormones. To give some idea of the capabilities of this system, at maximal parasympathetic stimulation the gland (1 g wet wt.) can produce a saliva flow of 300-700pl/min. The total protein concentration varies between 130 and 170mg/lOOml and the complex polysaccharide conjugates amount to 25-30mg/lOOml. Polyacrylamide-gel electrophoresis generally reveals a large number of protein components, some of which are ultrafiltrated serum components. Complementary staining with periodate/Schiff reagent shows at least two diffuse glycoconjugate-containing proteins. The composition of the sugar moiety synthesized by the gland has been the subject of much consideration. Dische et al. (1962) cannulated the dog submaxillary gland and elicited saliva by electrical stimulation and by intravenous injection of pilocarpine or acetylcholine. The minimally purified mucins obtained by these agents were each analysed for sugars, and it was found that, depending on the type and degree of stimulation, the ratio of terminating sugar residues expressed as sialate/fucose could vary
Vol. 5
BIOCHEMICAL SOCIETY TRANSACTIONS
holocrine secretion is not restricted to the well-known sebaceous glands. It is difficult to discern between the content of whole disintegrated cells and the mature product liberated from viable cells by secretion. The extramural glands of the tracts do not raise such difficulties. Although the structural aspects and kinetics of secretion have become rather well known, the underlying mechanism of membrane fusion and its regulation are by no means understood. The key to a better understanding might be found in the study of fusing membranes. The use of cultures of free secretory cells seems to be advantageous. The study of the conditions that favour fusions between cellular membranes, fragmentation vesicles and liposomes might also be expected to throw light on one of the most fundamental processes, controlling so many functions essential for organized life. Bauduin, H., Colin, M. & Dumont, J. E. (1969)Biochim. Biophys. Acta 174,722-733 Bock, E. & Jorgensen, 0. S. (1975)FEBSLett. 52,37-39 Bogart, B. J. (1975)J. Ultrastruct. Res. 52, 139-155 Bolender, R. P. (1974)J. Cell Biol. 61, 269-287 Davis, B. K. (1974)J. Reprod. Fertil. 41,241-244 de Camilli, P., Peluchetti, D. & Meldolesi, J. (1974)Nature (London) 248, 245-247 Geuze, J. J. & Kramer, M. F. (1974)Cell Tissue Res. 156, 1-20 Geuze, J. J., Kramer, M. F. & de Man, J. C. H. (1976) in Mammalian Cell Membranes (Jamieson, G . A. & Robinson, D. M., eds.), vol. 2,chapter 2,Buttenvorth, London Geuze, J. J. & Slot, J. W. (1976)Biol. Reprod. 15, 118-125 Harrop, T. 3. & Garrett, J. R. (1974)Cell Tissue Res. 154, 135-150 Jamieson, J. D.& Palade, G. E. (1971)J. Cell Biol. 48,503-522 Kim, S. K., Nasjleti, L. E. & Han, S. S. (1972)J. Ultrastruct. Res. 38, 371-389 Kramer, M. F. & Poort, C. (1972)J. CeN Biol.52, 147-158 Meldolesi, J. (1974)J. Cell Biol. 61, 1-13 Meldolesi, J. & Cova, D. (1972)J. Cell Biol. 55, 1-18 Meldolesi, J., Jamieson, J. D. & Palade, G. E. (1971)J. Cell Biol. 49, 130-158 Palade, G. E. & Bruns, R. R. (1968)J. Cell Biol. 37, 633-649 Raff, M., unpublished work, cited by Bretscher, M. S. (1976)Nature (London)261, 366-367 Satir, B., Schooley, C. & Satir, P. (1973)J. Cell Biol. 56, 153-176 Wallach, D.,Kirshner, N. & Schramm, M. (1975)Biochim. Biophys. Acta 375, 87-105 White, D. A. & Hawthorne, J. N. (1970)Biochem. J. 120,535-538 Winkler, H., Hortnagl, H., Rufener, C., Nakane, P. K. & Schneider, F. H. (1974) in CytopharmacologyofSecretion (Ceccarelli, B., Meldolesi, J. & Clementi, F., eds.), pp. 127-140, Raven Press, New York
Neurophysiological Control of Glycoprotein Secretion in the Tracheal Epithelium P. W. KENT* and J. G. WIDDICOMBEt *Glycoprotein Research Unit, University of Durham, Durham DH1 3LH, and t Department of Physiology, St. George’s Hospital Medical School, London S W17 OQT,U.K.
The formation of a mucus barrier, external to a mammalian epithelial tissue, is the outcome of two major processes, the biochemical pathways of glycoprotein assembly and the cellular physiology of secretion, i.e. glycoprotein storage and export. Though these processes are subject to their distinctive modes of regulation, stimulation and inhibition, they are inter-related by mechanisms which are as yet not understood. The tracheal epithelium is a promising tissue for the investigation of both processes. Apart from the obvious interest in a connexion with respiratory disorders, the innervation of the trachea is well known in a number of species and is accessible for experimental intervention. The tracheal epithelial mucus in the cat, rabbit, dog 1977
567th MEETING, DURHAM
41 5
Table 1. Carbohydrate composition of principal tracheal glycoproteins (jiractionated on DEAE-cellulose DE-52) Values are expressed as molar ratios relative to N-acetylglucosamine (=l); sialic acid is measured as N-acetylneuraminic acid. Rabbit*
Fucose Galactose N-Acetylglucosamine N-Acet ylgalactosamine Sialic acid Sulphate * From organ culture. t From tracheal perfusion.
Dog*
. (Soluble) (Insoluble) (Soluble) 0.84 0.37 0.23 1.03 0.92 1.60 1 .oo 1 .oo 1.oo 1.83 1 .oo 0.74 0.93 0.77 0.28
++
++
++
Catt
Gooset
(Soluble) (Soluble) 0.65 1.56 1 .oo 1.30 1 .oo 1 .oo 1.25 0.94 0.36 0.40 U
++
Table 2. Ratio of [35S]sulphate-and [3Hlglucose-derived radioactivity in cat tracheal mucins before and after administration of irritants Separate mucin collections were pooled as indicated. Controls (collections 1-5) represent the first five collections of radioactive mucin before addition of pilocarpine. Three fractions, collections (7), (8+9) and (12-1 5), were examined after pilocarpine treatment (collection 6). Control collections (lo), (11) and (16) represent mucin samples taken before and after the second phase of pilocarpine stimulation (collections 12-15). Pooled fraction (collections 17 and 18) was taken after NH, treatment. Irritant None (controls) Pilocarpine Pilocarpine Pilocarpine Pilocarpine None (controls) NH3
Collection (1-5)
(6) (7) (8,9) (12-15) (10, 11, 16) (17918)
Ratio 3H/35S 6.23 2.07 3.19 3.73 4.46 5.03 11.11
and in man is a mixed secretion arising from at least three cell types, namely epithelial goblet cells and mucous and serous cells of the submucosal glands. It has been of interest to explore whether these different cell types synthesize chemically different glycoproteins, and whether each is separately innervated, responding distinctively to irritants and stimulation. A connexion between glycoprotein secretion and nervous stimulation has long been known and there is some evidence of accompanying variations in chemical composition. This was first suggested by Dische et af. (1962) and Dische (1963), who showed that electrical or pilocarpine stimulation of dog submaxillary gland increased the fucose/sialic acid ratio. This has been further investigated (Winzler, 1973 ; Lombart & Winzler, 1975) as discussed by Phelps & Young (1977). The development of a new experimental approach has enabled mucus output to be measured by tracheal lavage of anaesthetized cats to which radioisotopic precursors, e.g. [35S]sulphate or [3H]glucose, had been administered into the blood or into the tracheal lumen (Gallagher et al., 1975). Sympathetic-nerve stimulation and sympatho-
Vol. 5
416
BIOCHEMICAL SOCIETY TRANSACTIONS
Table 3. Comparison of sugar composition of cat miicins obtained after administration of irritants Values were obtained by g.1.c. analysis of samples after ion-exchange fractionation on DE52 DEAE-cellulose. Results are expressed as molar ratios, relative to N-acetylglucosamine (=l). Treatment Mucus collections Sugars Fucose Galactose N-Acetylgalactosamine N-Acetylglucosamine Sialic acid
... ...
Pilocarpine (6)
Control
1.51 1.64 0.70
I .25 I .45 0.80
1 .oo
0.62
(1-5)
1 .oo 0.55
Ammonia (17 and 18) 0.12 1.15 0.65 I .oo 1.15
mimetic amines greatly enhanced output of 35S-labelledmucus above resting values. After either excitation the amount of label in the secretion rapidly returned to that of the resting state. These effects were prevented by 8-adrenergic blocking agents, e.g. propanolol, but not by a-blockade by using phentolamine. Stimulation of the parasympathetic nerve (vagal) also resulted in increased mucus output, of an order similar to that of the sympathetic effects. In this case, the effects were blocked by atropine, whereas pilocarpine, a parasympathomimetic agent, greatly increased the mucus outflow. Fractionation of the mucus glycoproteins on DEAE-cellulose DE52 and on Sephadex G-200 (Gallagher & Kent, 1975) showed two glycoproteins to be present, the electrophoretically less mobile fraction carrying 35S label. The composition of the material from the cat and other species is given in Table 1. Histological examination showed that most mucous and goblet cells produced a [35S]sulphate-labelled glycoprotein. Radioautography showed that pilocarpine and nervous stimulation caused release of labelled glycoprotein from submucosal glands (serous and mucous cells) rather than from epithelial goblet cells. Subsequent administration of NH3 to the same trachea promoted secretion from goblet cells (Gallagher et al., 1977). In a series of double-labelling experiments, Na235S04(1 mci) and ~-[l-~H]glucose (0.25mCi) were introduced, in medium, into the cat tracheal lumen. The KrebsHenseleit (1932) medium which filled the trachea was replaced hourly. The doublelabelled mucus attained a steady rate of formation after lhh. Pilocarpine (6.5pg/ml in medium) was given four times at intervals of 15 min. This was followed by two exposures to NH3 vapour (1 :loo) in air. After exposure to each stimulant the secreted mucus was washed from the trachea and examined chemically. The radioisotopic ratios are shown in Table 2 and the sugar composition of fractions is given in Table 3. It is evident that pilocarpine evokes a more highly sulphated much from submucosal glandular cells. Repeated pilocarpine stimulation results in diminished sulphation without pronounced effect on molecular size. The glycoprotein released from goblet cells by NH3 is distinctive in having a low sulphate content and high sialic acid. The cellular origins of mucus in the goose trachea are quite different from those of mammals in that only goblet cells are present. The ~ U C O L I secretion S here was stimulated by acetylcholine, an effect which was completely blocked by atropine. Neither a- nor &stimulant sympathomimetic amines affected the rate of secretion. Examination of the goose tracheal glycoproteins after fractionation by gel filtration revealed that fucose was absent but that mannose was consistently present (Phipps et al., 1976). The results suggest that in mammals the tracheal mucus is a mixture of glycoproteins of different cellular origins, that nervous control of mucous and serous cells in bronchial glands is exerted through the parasympathetic system, and that the various cellular sites are differently capable of stimulation by chemical irritants. 1977
567th MEETING, DURHAM
417
Dische, Z. (1963) Ann. N. Y. Acad. Sci. 106, 259-270 Dische, Z., Pallavicini, C., Kavaski, H., Smirnow, N., Cizek, L. & Chien, S. (1962) Arch. Biochem. Biophys. 97,459-469
Gallagher, J. T. & Kent, P. W. (1975) Biochem. J. 148, 187-196 Gallagher, J. T., Kent, P. W., Passatore, M., Phipps, R. J. & Richardson, P. S. (1975) Proc. R. Soc. London, Ser. B 192,49-76
Gallagher, J. T., Kent, P. W., Phipps, R. J. & Richardson, P. S. (1977) in Mucus in Healrh and Disease (Parke, D. V. & Elstein, M., eds.), Plenum, London, in the press Krebs, H. A. & Henseleit, K. (1932) Hoppe-Seyler’s 2. Physiol. Chem. 210, 33-66 Lornbart, C . G . & Winzler, R. J. (1975) Eur. J. Biochem. 49, 77-86 Phelps, C. F. & Young, A. M. (1977) Biochem. Soc. Trans. 5, 417-419 Phipps, R. J., Richardson, P. S . , Corfield, A., Gallagher, J. T., Jeffery, P. K., Kent, P. W. & Passatore, M. (1976) Philos. Trans. R. Soc. London, Ser. B in the press Winzler, R. J. (1973) in Membrane Mediated Information (Kent, P. W., ed.), vol. 1, pp. 3-19, Medical and Technical Publishing Co., Lancaster
The Regulation of Mucin Production in the Feline Submaxillary Gland CHARLES F. PHELPS and AILEEN M. YOUNG Department of Biological Sciences, University of Lancaster, Lancaster LA1 4 YQ, U.K.
The submaxillary gland offers a complex system in which to study the control of epithelial mucin secretion. This complexity has a number of roots. Apart from the large number of different animals whose submaxillary glands and their contents have been studied, each gland can exist in a gradation of functioning states between the wholly quiescent and the fully active. This activity may be promoted by sympathetic or parasympathetic innervation as well as by chemical means; the components produced by the gland are many and diverse, and include water, inorganic electrolytes, enzymes and growth-promoting factors as well as complex glycoproteins. Though information exists on many of the components mentioned above, the integrated knowledge of the functioning of the gland is largely fragmentary. For this reason we decided to study a preparation in vivo of cat submaxillary gland. The surgical procedure exposes one gland and enables cannulation of the duct, and also of the blood supply to the gland. Both sympathetic and parasympathetic innervation are exposed, allowing external experimental control to be exercised over the saliva flow. The contralateral gland that was merely exposed acted as control. With this experimental protocol we have a system in which: (a) there is production of a few characterizable glycoproteins; (b) the production is variable in quality and quantity, depending on degree and type of stimulus; ( c ) the preparation is isolatable from other humoral factors such as hormones. To give some idea of the capabilities of this system, at maximal parasympathetic stimulation the gland (1 g wet wt.) can produce a saliva flow of 300-700pl/min. The total protein concentration varies between 130 and 170mg/lOOml and the complex polysaccharide conjugates amount to 25-30mg/lOOml. Polyacrylamide-gel electrophoresis generally reveals a large number of protein components, some of which are ultrafiltrated serum components. Complementary staining with periodate/Schiff reagent shows at least two diffuse glycoconjugate-containing proteins. The composition of the sugar moiety synthesized by the gland has been the subject of much consideration. Dische et al. (1962) cannulated the dog submaxillary gland and elicited saliva by electrical stimulation and by intravenous injection of pilocarpine or acetylcholine. The minimally purified mucins obtained by these agents were each analysed for sugars, and it was found that, depending on the type and degree of stimulation, the ratio of terminating sugar residues expressed as sialate/fucose could vary
Vol. 5
