Morphological changes and EGF expression in the granular convoluted tubule cells of submandibular glands of Trypanosoma cruzi infected rats

We have previously demonstrated in rats that Chagas' disease affects the salivary glands, by promoting an enlargement of the submandibular gland. In order to further investigate possible functional alterations on infected submandibular glands, the objective of the present study was to analyze epidermal growth factor (EGF) expression on rat submandibular glands during Trypanosoma cruzi infection. Results demonstrated that infected rats presented lower levels of testosterone, and morphological changes in the granular convoluted tubule (GCT) cells of the submandibular glands, along with acinar enlargement and delayed ductal maturation at the developing granular ducts. Immunohistochemistry analysis additionally showed that only few cells immunolabelled with anti-EGF on infected rats during the acute phase of Chagas' disease, while after 64 and 90 days (chronic phase) of infection, EGF expression was similar to non-infected rats. The present findings suggest that at the acute phase of Chagas' disease, lower levels of testosterone may lead to a delayed maturation of GCT, which positively correlates with decreased EGF production by submandibular glands cells.     

Changes with senescence in the fine structure of the granular convoluted tubule of the submandibular gland of the mouse

Cells of the granular convoluted tubules (GCTs) of the submandibular gland of senescent male mice show structural changes indicative of functional decline. In order to define the nature of these age-related changes more clearly, the fine structure of GCT cells of 12- and 28-month-old males was compared. In old mice, there was cell-to-cell variation in the extent of these changes, with some cells of senescent males appearing no different from those of young adults. In affected cells the most striking alterations were seen in secretion granules and lysosomal elements. Secretion granules varied greatly in size, with some GCT cells having only very fine apical granules. Secondary lysosomes and large lipofuscin granules were frequent in the basal cytoplasm. Very large dense bodies (3-5 micron) occurred in many cells. These possibly represent intracellular pools of released secretory materials, as they were occasionally seen in continuity with the luminal contents. Structures whose appearance was intermediate between the very large dense bodies and lipofuscin granules were common, suggesting crinophagic activity. There was an apparent decrease in numbers of polysomes and in the extent of the Golgi apparatus. These fine structural changes are consistent with impairments with advanced age in synthesis and posttranslational processing of secretory products by affected GCT cells. In addition to cell-to-cell variation in any one male, there was also interanimal variation in the degree and extent of these senescent changes.     

Thyroxine accelerates the differentiation of granular convoluted tubule cells and the appearance of epidermal growth factor in the submandibular gland of the neonatal mouse

Summary The effects of the administration of thyroxine (T4) on the postnatal cytodifferentiation of granular convoluted tubule (GCT) cells of the submandibular gland (SMG) of Lewiss-Webster mice were studied by light and electron microscopy. From birth, mice of both sexes were injected daily with T4 (sc 0.4 g/g BW) and were sacrificed 24 h after the last injection at 7, 9, 11, 14 and 21 days of age. Control mice received vehicle only. In control mice, granulated striated duct (SD) cells were first detected at 9 days and 7 days of age by light- and electron microscopy, respectively. Furthermore, a few scattered granulated SD cells were observed by light microscopy as early as day 7 in T4-treated mice of both sexes. At 21 days of age, in mice given T4, GCT cells were larger and more numerous and the Golgi apparatus, rough endoplasmic reticulum, and secretion granules were more abundant. In control mice, immunocytochemical staining for epidermal growth factor-(EGF) was first detectable at day 21 at the light- and electron-microscopic levels. However, positively stained cells were first observed in T4-treated mice by light- and electron-microscopic immunocytochemistry at 14 and 11 days of age, respectively. Moreover, in the 21-day-old T4-treated mice, the number of immunoreactive GCT cells, as well as the intensity of the staining per cell, was markedly increased as compared to controls. EGF immunostaining was restricted to GCT cells, and by immuno-electron-microscopy was only seen in apical secretory granules in granulated SD cells and GCT cells. There were no sex differences in the differentiation of the duct system under any conditions. It is concluded that T4 stimulates the biosynthesis of EGF by an acceleration of the differentiation of the GCT precursor cells to mature cells.Supported in part by grant no. MT-5730 from the Medical Research Council of CanadaHolder of a fellowship from the Medical Research Council of CanadaScholar of the Fonds de la Recherche en Santé du Québec     

Proline-rich-protein promoters direct LacZ expression to the granular convoluted tubular cells of the submandibular gland in adult transgenic mice

The ability of two mouse PRP gene promoters to direct the expression of the bacterial lacZ reporter gene was tested in transgenic mice. Transgenes A1-lacZ and C1-lacZ consisted of 8.2 kb A1 and 7.8 kb C1 PRP promoters respectively fused to the lacZ coding sequence. A1 and C1 are two A-type PRP genes isolated from the inbred SWR mice, which show the same gene structure and similar sequence to the closely related MP2 and M14 PRP genes previously cloned from outbred CD-1 mice. We here show that both A1-lacZ and C1-lacZ transgenes have very similar expression patterns: (1) they expressed the lacZ gene in all 14 established transgenic lines under normal (non-stimulated) conditions; (2) the expression was restricted to the granular convoluted tubular cells of the submandibular glands; (3) the expression was developmentally regulated beginning at sexual maturation and lasting to at least 1.5 years of age; and (4) expression in some lines was probably influenced by sex hormones, since higher expression was found in males than in females. A1-lacZ and C1- lacZ are the first transgenes derived from the PRP/GRP (glutamine/glutamic acid-rich protein) gene superfamily to be expressed in the granular convoluted tubular cells (with known endocrine functions), rather than in the acinar cells (with mainly exocrine functions) of the submandibular glands     

Cytodifferentiation of striated duct cells and secretory cells of the convoluted granular tubules of the rat submandibular gland.

The structural and functional development of the striated ducts and convoluted granular tubules (CGT) of the rat submandibular gland (SMG) were studied by electron microscopy and alkaline protease chemistry. Development of the SMG was followed from 14 days of gestation through 30 weeks of age. The specialized morphology of the basal aspect of the striated duct cells arises from cellular extensions which are first seen at 20 days of gestation. These processes elongate and intertwine with similar processes from adjacent cells, and as the cells enlarge the processes are compressed together giving the appearance of "infolding" of the basal plasma membrane. Mitochondria migrate to the basal part of the cell and are seen in close relationship to the cellular extensions throughout the development of these cells. Development of the striated duct is complete by one week after birth. The CGT develop from the proximal portions of intralobular striated ducts. At one week after birth, cells of the proximal striated duct demonstrate apical vacuoles. By two weeks after birth these vacuoles are replaced by distinct zymogen-like granules. There is a progressive accumulation of large numbers of secretory granules in the CGT cells as the animals age. However, rough endoplasmic reticulum is a relatively inconspicuous cellular component throughout development. The accumulation of alkaline protease activity in the gland closely parallels the pattern of granule accumulation.     

Developmental and androgenic regulation of the immunocytochemical distribution of mK1, a true tissue kallikrein, in the granular convoluted tubule of the mouse submandibular gland.

The action of androgens on the immunocytochemical distribution of mK1, a true tissue kallikrein, was examined in the submandibular gland (SMG) of developing and adult mice by indirect enzyme-labeled and immunogold-labeled antibody methods for light and electron microscopy, respectively. In both sexes at 3 weeks of age, essentially all of the immature granular convoluted tubule (GCT) cells were uniformly immunostained. At 4 weeks of age (the onset of puberty), morphological differences between the two sexes appeared in the GCTs, in which some cells became immunonegative. Thereafter, the immunonegative GCT cells became more abundant in the SMG of males than of females and considerable intercellular variation in staining intensity for mK1 was seen, especially in males. A few slender GCT cells with strong immunoreactivity appeared in GCT segments only in males. Castration of males resulted in an increase in the number of immunopositive GCT cells, whereas administration of dihydrotestosterone (DHT) decreased the number of immunopositive GCT cells in the SMGs of both sexes. Slender GCT cells immunoreactive for mK1 were seen in females treated with DHT for 6 days. However, there were no immunostained slender GCT cells in female SMGs after injection of DHT for 2 weeks. Immunoelectron microscopy disclosed this type of cell in male SMGs, which closely resembles immature GCT cells of prepubertal mice, with a few small secretory granules uniformly labeled with gold particles, a sparse Golgi apparatus and RER, and basal infoldings. In mature male SMGs and in SMGs of DHT-treated females and castrated males, typical GCT cells had a well-developed Golgi apparatus and a net-like RER but few to no basal infoldings, whereas in the female gland equivalent cells had moderately developed RER and some basal infoldings. These results suggest that mK1 is one of the enzymes characteristically present in immature GCT cells and that its synthesis is inhibited in part by androgens, resulting in decreased numbers of immunopositive cells.     

Effect of testosterone on the amount of serous-like granules in convoluted tubular cells of mouse submandibular glands.

The effect of testosterone on the amount of granules present in convoluted tubular cells of the submadibular glands of mice was studied by the following two methods; 1) an immunochemical method using antiserum specific to the granular components, and 2) a histographic method. The results obtained by these two methods were in agreement. The amounts of the granules in normal female and castrated male mice were one-tenth to one-twentieth of that in normal male mice. When male mice were castrated, the amount of granules decreased, reaching a minimum 20 days after the aperation the injection of the male hormone, testosterone, into castrated male mice caused an increase in the amount of granules; this increase reached a maximum 15 days after the injection. The increase of granules caused by testosterone injection was almost completely prevented by inhibitors of protein synthesis, actinomycin D and puromycin. This suggests that protein synthesis was indispensable to the increase in the amount of granules. In male mice, the injection of female hormones scarcely affected the amount of granules. Kinetic analysis of the decrease and increase of granules on castration and testosterone injection suggested that the male hormone stimulated granule synthesis, but it hardly influenced the loss of granules.     

Postnatal developmental changes in submandibular glands of rats and mice

The submandibular glands of mice and rats are not fully developed at birth. In early postnatal life, differentiation of acini takes place before that of granular convoluted tubule (GCT) cells. The latter develop from striated duct cells, and first appear in both species around 15 days of age. In mice their full development gets under way by 20 days of age and is rapid in males and slow in females, resulting in a clear sexual dimorphism in adults. In rats, GCT development is more protracted, and accelerates around 40 days of age, with no sexual dimorphism seen at any time. The course of postnatal development of several GCT cell products is correlated with the cytodifferentiation of these cells. Reliable data are available for the development of amylase, proteases (including kallikrein), renin, epidermal growth factor, and nerve growth factor. Preliminary information exists for a glucagon-like substance. Cytodifferentiation of GCT cells is under hormonal control. Androgens alone can not precociously induce GCT cells, but thyroid hormones can do so, acting either alone or synergistically with androgens.     

Epidermal growth factor in serum, urine, submandibular glands and kidneys of diabetic mice

Levels of epidermal growth factor (EGF) in serum were significantly decreased in streptozotocin (STZ)-diabetic mice (446 +/- 168 pg/ml after 1 week and 423 +/- 52 after 4 weeks vs 766 +/- 162 pg/ml in controls, P.002 and less than .001. respectively) and in genetically diabetic ob/ob mice (455 +/- 285 vs 962 +/- 453 pg/ml in nondiabetic ob/+ controls, P.043). The urinary excretion of EGF was significantly increased in STZ mice (104 +/- 53 vs 51 +/- 23 ng/h, P.013) but unchanged in ob/ob mice (33 +/- 9 vs 45 +/- 16 ng/h, P.134). However, when expressed per mg creatinine it was decreased in both cases: in STZ mice to 680 +/- 250 ng/mg at 1 week and 684 +/- 211 at 4 weeks vs 1250 +/- 303 ng/mg in controls (P less than .01); and in the ob/ob mice to 552 +/- 117 vs 1237 +/- 300 ng/mg in ob/+ controls (P less than .01). EGF content of the submandibular glands of STZ mice remained unchanged at 1 week (13.1 +/- 2.9 vs 11.0 +/- 1.8 micrograms/mg protein, P.170) but dropped by 4 weeks (4.7 +/- 1.2 micrograms/mg, P less than .001); in the ob/ob mice it was less than 20% that of controls (2.1 +/- 0.8 vs 12.2 +/- 3.6 micrograms/mg protein). In kidneys, the EGF content was not altered in either ob/ob (524 +/- 50 vs 571 +/- 33 pg/mg protein) or STZ mice (652 +/- 183 vs 665 +/- 80 pg/mg). The preproEGF mRNA level in STZ-treated mice was reduced after 4 weeks in submandibular glands but not in kidneys. The results show that diabetes affects EGF production, utilization and/or excretion in mice and that kidneys are spared from suppression of EGF synthesis that is pronounced in the submandibular glands.     

Oscillatory changes in cells of the submandibular glands in mice during the secretory cycle]

By means of 3H-leucine radioautography, ultrastructural and morphometrical analysis, it has been demonstrated that in the cells of the acinar (Ac) and granular (Gr) parts of the submandibular gland (SMG) in mice with a synchronized for 3 h nutrition cycle there are endogenic fluctuations (for about 1 h) of the secretory process. They are demonstrated as alterations in intensity of protein synthesis in the cells, their areas and ultrastructure. In the Ac cells at the moment of feeding and in 50, 110, 150 min after feeding maximal leucine incorporation is observed. The changes in the cell area during the first hour occurs with 10 minutes' overtake of the incorporation intensity, and during the following 2 h an equilibrium is reached between these two parameters, when the maximal contents of the label corresponds to the minimal area of the cells. In the Gr cells one maximum of 3H-leucine incorporation occurs in 60-80 min after feeding. The minimal content of the label takes place in 10 and 120 min after feeding. During the time mentioned inverse ratio between the intensity incorporation of the labelled isotope and the change of the cell volume is observed. This is connected with autoregulatory processes in the cells. It is possible that in the Ac cells together with removal of the secretory granules by means of exocytosis (in 10-20 min after feeding) the secretory material is removed before it is formed as granules by parvivesicular extrusion. In the Gr cells apocrinic and endocrinic secretion takes place. Presence of certain specific formations (secretory lysosomes) explains peculiarities of the secretory process in the SMG.     

Invaginated apical vacuoles in the cells of the proximal convoluted tubule in the rat kidney

Summary Following perfusion fixation of the rat kidney with glutaraldehyde the proximal tubule cells display small apical vacuoles, large apical vacuoles, and apical vacuoles in which a part of the limiting membrane is invaginated into the vacuole. These invaginated apical vacuoles occur more frequently in proximal convoluted tubules than in proximal straight tubules. One tubular cell may contain apical vacuoles of different sizes and stages of invagination, ranging from larger vacuoles with a wide lumen and a small area of invaginated membrane to smaller elements with no apparent lumen and a large area of invaginated membrane. Invaginated apical vacuoles lie either singly in the cytoplasm or close to the membranes of other apical vacuoles, but never in contact with the cell membrane or the membranes of lysosomes, endoplasmic reticulum, Golgi apparatus, mitochondria and peroxisomes.These findings suggest that the invaginated apical vacuoles are not fixation artifacts, but rather develop in living state in cells of the proximal tubule from spherical endocytotic elements.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Ultrastructural localization of glucose-6-phosphatase activity in proximal convoluted tubule cells of rat kidney

Summary The ultrastructural localization of glucose 6-phosphatase activity was investigated in the proximal convoluted tubule cells of the rat kidney. The reaction product for the enzyme activity was present in the endoplasmic reticulum and nuclear envelope, as reported for the hepatic enzyme and others, but was absent from the brush border, plasma membrane and other organelles. The metabolic significance of the association of this enzyme with the endoplasmic reticulum and the role of the enzyme in the active reabsorption and transport of glucose in the renal tubules are discussed.     

Identification of gene expression profile of neural crest-derived cells isolated from submandibular glands of adult mice

Neural crest cells in the embryo migrate to reach target sites as neural crest-derived cells (NCDCs) where they differentiate into a variety of derivatives. Some NCDCs are maintained in an undifferentiated state throughout the life of the animal and are considered to be a useful cell source for regenerative medicine. However, no established method to obtain NCDCs sufficient for regenerative medicine from adults with high purity has been presented, since their distribution in adult tissues is not fully understood. It is critical to identify reliable markers for NCDCs in adults, as the expressions of P0 and Wnt1, the most reliable NCDC markers, are shut off in the embryonic stage. To analyze the characteristics of NCDCs in adult tissues, we utilized a double transgenic mouse strain, P0-Cre/CAG-CAT-EGFP transgenic mice (P0 mice), in which NCDCs were shown to express EGFP and we were able to recognize GFP-positive cells in those. We focused on the submandibular glands (SMGs), which are known to be derived from the neural crest. GFP-positive cells were shown to be scattered like islands in the SMGs of adult P0 mice. We surgically removed SMGs from adult mice and digested samples into single cell suspensions. GFP-positive cells separated using flow cytometry expressed a high level of Sox10, a marker of embryonic neural crest cells, suggesting successful isolation of NCDCs. To identify candidate marker genes in isolated NCDCs, we performed DNA microarray analyses and real-time PCR analysis of GFP-positive and -negative cells isolated from P0 mice, then selected genes showing differential gene expression patterns. As compared to GFP-negative cells, GFP-positive cells expressed Gpr4 and Ednrb at higher levels, whereas Pdgfra and Pdgfrb were expressed at lower levels. Furthermore, DNA microarray analysis showed that GFP-positive cells were positive for aquaporin 5, a marker for acinar cells. Together, our results indicate that NCDCs in adult SMGs have characteristic gene expression profiles specially their cell surface molecules. Cell sorting using a combination of these specific cell surface proteins would be a useful strategy for isolation of NCDCs from SMGs with high purity.     

Characterization of glucose transport by cultured rabbit kidney proximal convoluted and proximal straight tubule cells

Rabbit kidney proximal convoluted tubule (RPCT) and proximal straight tubule (RPST) cells were independently isolated and cultured. The kinetics of the sodium-dependent glucose transport was characterized by determining the uptake of the glucose analog alpha-methylglucopyranoside. Cell culture and assay conditions used in these experiments were based on previous experiments conducted on the renal cell line derived from the whole kidney of the Yorkshire pig (LLC-PK1). Results indicated the presence of two distinct sodium-dependent glucose transporters in rabbit renal cells: a relatively high-capacity, low-affinity transporter (V(max) = 2.28 +/- 0.099 nmoles/mg protein min, Km = 4.1 +/- 0.27 mM) in RPCT cells and a low-capacity, high-affinity transporter (V(max) = 0.45 +/- 0.076 nmoles/mg protein min, K(m) = 1.7 +/- 0.43 mM) in RPST cells. A relatively high-capacity, low-affinity transporter (V(max) = 1.68 +/- 0.215 nmoles/mg protein min, Km = 4.9 +/- 0.23 mM) was characterized in LLC-PK1 cells. Phlorizin inhibited the uptake of alpha-methylglucopyranoside in proximal convoluted, proximal straight, and LLC-PK1 cells by 90, 50, and 90%, respectively. Sodium-dependent glucose transport in all three cell types was specific for hexoses. These data are consistent with the kinetic heterogeneity of sodium-dependent glucose transport in the S1-S2 and S3 segments of the mammalian renal proximal tubule. The RPCT-RPST cultured cell model is novel, and this is the first report of sodium-dependent glucose transport characterization in primary cultures of proximal straight tubule cells. Our results support the use of cultured monolayers of RPCT and RPST cells as a model system to evaluate segment-specific differences in these renal cell types.     

Protein and DNA levels in the submandibular salivary glands of isoproterenol stimulated mice

The DNA and soluble and sedimental protein levels were studied in isoproterenol stimulated mouse submandibular salivary glands over a period of 200 h. Hypertrophy, the predominant cellular phenomenon following chronic isoproterenol treatment, was associated with a marked elevation of 14C-leucine incorporation into the buffer soluble protein fraction and a less pronounced isotope incorporation into the sedimental sub-cellular fraction as well as continued DNA synthesis. These findings are contrary to the accepted definitions of hypertrophy which preclude continued DNA synthesis. A suggestion is made, therefore, that in the system used the polyploidy associated with the hypertrophy involves.