Acta hi8tcehom. Bd. 58, S. 63-6i (1977)
Department of Histology, Institute of Medical Biology, Medical School, Gdansk, Poland
The effect of ACTH and cortisone on the behaviour of arylsulphatases in the salivary glands of white rat By ALICJA KOSTULAK With plate I (Received ;\Iarch 26, 1976)
Summary The effect of ACTH and cortisone on the behaviour of arylsulphatases in the sublingual and submaxillary glands was studied by means of histochemical methods. A distinct increase in enzymatic activity in the secretory cells of salivary glands was observed.
Many reports show (DAVIDOFF 1973, HooK et al. 1970, KOSTULAK 1974, MILSOM 1970, THYBERG 1972) that arylsulphatases are widely present in the tissues of many animals, and a high activity has been observed, among other things, in secretory epithelia (Hsu and TAPPEL 1965, KOSTULAK 1975, MAKITA and SANDBORN 1971). The occurrence of these enzymes some authors connect with the metabolism of sulphated polysaccharides (DoGSON 1968, MARTIN et al. 1969, MEYER 1969). However, although the specificity and intracellular localization of these enzymes in different tissues have been described previously, there are only a few reports about their localization in the salivary gland, and the functional role of arylsulphatases in the physiological function of the salivary glands. The observations reported by numerous authors (KOZLOWSKA 1973, KYAW and MELLORS 1972, SACHS and DE DUVE 1962), and in ours studies (KOSTULAK 1975) indicate that the activity of lysosomal enzymes, including arylsulphatases, is modified by steroid hormones. Moreover, it is known that these hormones have an influence on the composition and nature of the carbohydrate-protein complexes produced by the salivary glands (KOFOED et al. 1973, KOSTULAK et al. 1972). Therefore it seemed interesting to examine the behaviour of arylsolphatases in tho salivary glands after treatment with large doses of ACTH and cortisone, and find if there is any correlation between the activity of these enzymes and the changes of sulphated polysaccharides observed previously in the cell of the salivary glands after applying ACTH and cortisone.
Material and methods The experiments were carried out with 30 male rats (Wistar) sexually mature, weighing about 200 g each.
64
ALIOJ A KOSTULAK
The animals were divided into 3 equal groups. The 1st group consisted of control rats. In the 2nd group the rats were injected intraperitoneally with 25 mg of cortisone acetate (N. V. Organon, OOS, Holland) 5 times at 2 h intervals. The animals of the 3rd group were treated with ACTH Laboratoires Byla, Paris), using the same dose levels, and following the same scheme as that for the animals of the 2nd group. Throughout the experimental period the animals had acces to drinking water but received no food. 90 min after the last injection the animals were anaesthetized with ether, and fragments of the sublingual and submaxillary glands were taken for examination. The activity of arylsuphatase was determined by the method of RUTENBURG et al. 1952, using potassium 6-bromo-2-naphthylsulfate (Da}ac) as a substrate. The specificity of the enzyme reaction was demonstrated by incubating the sections in a medium free of substrate, and in the complete medium, after treating them with heat (90 °0 for 5 min).
Results Su blingual gland In the control the acinar cells showed a granular reaction localized in the basal cytoplasm. The semilunar cells showed a higher enzymatic activity than that of the acinar cells, and the reaction was uniformly distributed throughout their cytoplasm (Fig. 1). Highest enzymatic reaction, localized in the basal cytoplasm, was found in the duct cells. In the experimental groups of rats the enzymatic activity increased, mainly in the acinar cells, and these changes were more visible after applying cortisone (Fig. 2). Su bmaxillary gland In the control group the acinar cells showed fine-granular reaction, the reaction being uniformly distributed throughout their cytoplasm. In the cell of granular tubules the activity of the enzyme was much weaker, localized mainly in the basal cytoplasm along the cell membranes (Fig. 3). The excretory duct cells showed the same localization of the enzyme, the enzymatic reaction in them being highest. In the group of animals treated with hormones the enzymatic reaction was slightly diminished in the acinar cells, whereas in the cell of granular tubules the enzymatic activity distinct increased, particularly after the application of cortisone (Fig. 4). As compared with the control animals, no changes were observed in the excretory duct cells. Discussion Sulphates are a known constituent of the polysaccharide prosthetic fraction of salivary gland mucus, and sulphated glycoproteids were isolated from human and canine saliva (KROGSTAD et al. 1974, LOMBARD and WINZLER 1972, MARTIN et al. 1969, PANKSTAFF and DE BORURNE 1974). In the sublingual gland sulphated polysaccharides were demonstrated by histochemical and autographic methods (KOFOED et al. 1969, KOSTULAK 1975, LEPPI et al. 1968). As indicated by most studies (ENOMOTO and SCOTT 1969, RAVETTO et al. 1966), the amount of sulphated polysaccharides in t.he submaxillary gland is small, but attention is drawn to the presence of sulpholipids (PITCHARD
The effect of ACTH and cortisone
65
and RUSEN 1972) which provide another, in addition to sulphated polysaccharides, natural substrate for arylsulphatases. GUTTLER 1971 stressed the considerable contribution also of sulpllOlipids to the modifying mechanism of regulation of intracellular enzymes including arylsulphatases. As indicated by a number of cytochemical and u]t,rastructural investigations (DAVIDOFF and GALABOV 1973, HOOKET et al. 1970, LEZNICKI and BLESZYNSKI 1970,MAKITA and SANDBORN 1971), arylsulphatases are mostly lysosomal enzymes, although their presence was demonstrated also in other cell structures (M"C"RATA et al. 1975, RAPPAY et al. 1973), e. g., microsomes (MILSOM 1970), mitochondria (MAKITA 1971), GOLGI apparatus (DAVIDOFF 1973, THYBERG 1972). This variable location probably depends on the type of arylsulphatase described, for it has been known that connected with tho lysosomal fraction are primarily arylsulphatase A and B, whereas arylsulphatase C is related to the microsomal fraction. The investigations carried out so far do not permit a more exact determination of the intracellular localization of the enzymes discussed. On the basis of the results obtained it may be stated that the hormones used cause a clear increase in the activity of arylsulphatases in the salivary glands studies. However, it must be stressed that the enzymic activity and the reactivity in relation to the hormones used vary with the cell types. This seems to be determined by the fact that in the salivary glands each type of secretory cell has it own enzyme set which reacts to the hormones in a different way, and is closely correlated with the composition and chemical structure of the polysaccharides present in the particular cell type. In our investigations a clearly higher reactivity in relation to the hormones used was found for acinar cell arylsulphatases in both salivary glands. These cells are known to participate most actively in the production of protein-carbohydrate complexes present in the salivary mucus produced. In his studios JHONSON (1974) also emphasizes the role of lysosomal enzymes in the regulation of the content of secretory material in the salivary glands. In the present studies the effect of cortisone on the activity of the enzymes studied was more marked, which may be accounted for by a damaging action of this enzyme on the membranes, thus increasing their permeability to the substrate. Cortisone is know (KYAW and MELLORS 1972, WEBER et al. 1961) to selectively cause an increase in the activity of lysosomal enzymes involved in the metabolism of saccharide complexes, and in this way the specificity may be expressed of a hormonal effect at the enzymic level. A number of earlier studies (BOVERS and DE D"C"VE 1967, KOZLOWSKA 1973, KYAW 1972) indicate that steroid hornones modify the activity of arylsulphatases, but these findings do not always agree with reported by other authors. BOVERS and DE DUVE (1976) observed a lowered enzymic activity in lymphocytes, following treatment with cortisone. Studies carried out by KYAWA (1972) and other investigators (KOSTULAK 1975, KOZLOWSKA 1973, SACHS and DE DUVE 1962) indicated an increased activity of these enzymes in various organs, due to the application of steroid hormones. The discrepancy described may result from the use of different experimental conditions, prolonged and short treatment with hormones. Our earlier observation of the effect of ACTH and cortisone on the carbohydrate-protein complexes of salivary glands (K
Department of Histology, Institute of Medical Biology, Medical School, Gdansk, Poland
The effect of ACTH and cortisone on the behaviour of arylsulphatases in the salivary glands of white rat By ALICJA KOSTULAK With plate I (Received ;\Iarch 26, 1976)
Summary The effect of ACTH and cortisone on the behaviour of arylsulphatases in the sublingual and submaxillary glands was studied by means of histochemical methods. A distinct increase in enzymatic activity in the secretory cells of salivary glands was observed.
Many reports show (DAVIDOFF 1973, HooK et al. 1970, KOSTULAK 1974, MILSOM 1970, THYBERG 1972) that arylsulphatases are widely present in the tissues of many animals, and a high activity has been observed, among other things, in secretory epithelia (Hsu and TAPPEL 1965, KOSTULAK 1975, MAKITA and SANDBORN 1971). The occurrence of these enzymes some authors connect with the metabolism of sulphated polysaccharides (DoGSON 1968, MARTIN et al. 1969, MEYER 1969). However, although the specificity and intracellular localization of these enzymes in different tissues have been described previously, there are only a few reports about their localization in the salivary gland, and the functional role of arylsulphatases in the physiological function of the salivary glands. The observations reported by numerous authors (KOZLOWSKA 1973, KYAW and MELLORS 1972, SACHS and DE DUVE 1962), and in ours studies (KOSTULAK 1975) indicate that the activity of lysosomal enzymes, including arylsulphatases, is modified by steroid hormones. Moreover, it is known that these hormones have an influence on the composition and nature of the carbohydrate-protein complexes produced by the salivary glands (KOFOED et al. 1973, KOSTULAK et al. 1972). Therefore it seemed interesting to examine the behaviour of arylsolphatases in tho salivary glands after treatment with large doses of ACTH and cortisone, and find if there is any correlation between the activity of these enzymes and the changes of sulphated polysaccharides observed previously in the cell of the salivary glands after applying ACTH and cortisone.
Material and methods The experiments were carried out with 30 male rats (Wistar) sexually mature, weighing about 200 g each.
64
ALIOJ A KOSTULAK
The animals were divided into 3 equal groups. The 1st group consisted of control rats. In the 2nd group the rats were injected intraperitoneally with 25 mg of cortisone acetate (N. V. Organon, OOS, Holland) 5 times at 2 h intervals. The animals of the 3rd group were treated with ACTH Laboratoires Byla, Paris), using the same dose levels, and following the same scheme as that for the animals of the 2nd group. Throughout the experimental period the animals had acces to drinking water but received no food. 90 min after the last injection the animals were anaesthetized with ether, and fragments of the sublingual and submaxillary glands were taken for examination. The activity of arylsuphatase was determined by the method of RUTENBURG et al. 1952, using potassium 6-bromo-2-naphthylsulfate (Da}ac) as a substrate. The specificity of the enzyme reaction was demonstrated by incubating the sections in a medium free of substrate, and in the complete medium, after treating them with heat (90 °0 for 5 min).
Results Su blingual gland In the control the acinar cells showed a granular reaction localized in the basal cytoplasm. The semilunar cells showed a higher enzymatic activity than that of the acinar cells, and the reaction was uniformly distributed throughout their cytoplasm (Fig. 1). Highest enzymatic reaction, localized in the basal cytoplasm, was found in the duct cells. In the experimental groups of rats the enzymatic activity increased, mainly in the acinar cells, and these changes were more visible after applying cortisone (Fig. 2). Su bmaxillary gland In the control group the acinar cells showed fine-granular reaction, the reaction being uniformly distributed throughout their cytoplasm. In the cell of granular tubules the activity of the enzyme was much weaker, localized mainly in the basal cytoplasm along the cell membranes (Fig. 3). The excretory duct cells showed the same localization of the enzyme, the enzymatic reaction in them being highest. In the group of animals treated with hormones the enzymatic reaction was slightly diminished in the acinar cells, whereas in the cell of granular tubules the enzymatic activity distinct increased, particularly after the application of cortisone (Fig. 4). As compared with the control animals, no changes were observed in the excretory duct cells. Discussion Sulphates are a known constituent of the polysaccharide prosthetic fraction of salivary gland mucus, and sulphated glycoproteids were isolated from human and canine saliva (KROGSTAD et al. 1974, LOMBARD and WINZLER 1972, MARTIN et al. 1969, PANKSTAFF and DE BORURNE 1974). In the sublingual gland sulphated polysaccharides were demonstrated by histochemical and autographic methods (KOFOED et al. 1969, KOSTULAK 1975, LEPPI et al. 1968). As indicated by most studies (ENOMOTO and SCOTT 1969, RAVETTO et al. 1966), the amount of sulphated polysaccharides in t.he submaxillary gland is small, but attention is drawn to the presence of sulpholipids (PITCHARD
The effect of ACTH and cortisone
65
and RUSEN 1972) which provide another, in addition to sulphated polysaccharides, natural substrate for arylsulphatases. GUTTLER 1971 stressed the considerable contribution also of sulpllOlipids to the modifying mechanism of regulation of intracellular enzymes including arylsulphatases. As indicated by a number of cytochemical and u]t,rastructural investigations (DAVIDOFF and GALABOV 1973, HOOKET et al. 1970, LEZNICKI and BLESZYNSKI 1970,MAKITA and SANDBORN 1971), arylsulphatases are mostly lysosomal enzymes, although their presence was demonstrated also in other cell structures (M"C"RATA et al. 1975, RAPPAY et al. 1973), e. g., microsomes (MILSOM 1970), mitochondria (MAKITA 1971), GOLGI apparatus (DAVIDOFF 1973, THYBERG 1972). This variable location probably depends on the type of arylsulphatase described, for it has been known that connected with tho lysosomal fraction are primarily arylsulphatase A and B, whereas arylsulphatase C is related to the microsomal fraction. The investigations carried out so far do not permit a more exact determination of the intracellular localization of the enzymes discussed. On the basis of the results obtained it may be stated that the hormones used cause a clear increase in the activity of arylsulphatases in the salivary glands studies. However, it must be stressed that the enzymic activity and the reactivity in relation to the hormones used vary with the cell types. This seems to be determined by the fact that in the salivary glands each type of secretory cell has it own enzyme set which reacts to the hormones in a different way, and is closely correlated with the composition and chemical structure of the polysaccharides present in the particular cell type. In our investigations a clearly higher reactivity in relation to the hormones used was found for acinar cell arylsulphatases in both salivary glands. These cells are known to participate most actively in the production of protein-carbohydrate complexes present in the salivary mucus produced. In his studios JHONSON (1974) also emphasizes the role of lysosomal enzymes in the regulation of the content of secretory material in the salivary glands. In the present studies the effect of cortisone on the activity of the enzymes studied was more marked, which may be accounted for by a damaging action of this enzyme on the membranes, thus increasing their permeability to the substrate. Cortisone is know (KYAW and MELLORS 1972, WEBER et al. 1961) to selectively cause an increase in the activity of lysosomal enzymes involved in the metabolism of saccharide complexes, and in this way the specificity may be expressed of a hormonal effect at the enzymic level. A number of earlier studies (BOVERS and DE D"C"VE 1967, KOZLOWSKA 1973, KYAW 1972) indicate that steroid hornones modify the activity of arylsulphatases, but these findings do not always agree with reported by other authors. BOVERS and DE DUVE (1976) observed a lowered enzymic activity in lymphocytes, following treatment with cortisone. Studies carried out by KYAWA (1972) and other investigators (KOSTULAK 1975, KOZLOWSKA 1973, SACHS and DE DUVE 1962) indicated an increased activity of these enzymes in various organs, due to the application of steroid hormones. The discrepancy described may result from the use of different experimental conditions, prolonged and short treatment with hormones. Our earlier observation of the effect of ACTH and cortisone on the carbohydrate-protein complexes of salivary glands (K
