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Hypohidrotic (anhidrotic) ectodermal dysplasia (HED), the most common of the approximately 150 described ectodermal dysplasias, is a disorder characterized by abnormal hair, teeth, sweat glands, and salivary glands. Mutations in the EDA (ectodysplasin-A) and EDAR (ectodysplasin-A receptor) genes are responsible for X-linked and autosomal HED, respectively. Abnormal phenotypes similar to HED are seen in Tabby (Eda(Ta)) and downless (Edar(dl)) mutant mice. Although recent studies have focused on the role of Eda/Edar signaling during hair and tooth development, very little is known about its role during embryonic submandibular salivary gland (SMG) development. To this end, we analyzed the SMG phenotypes in Tabby (Ta) and downless (dl) mutant mice and determined that Ta SMGs are hypoplastic, whereas dl SMGs are severely dysplastic. The absence of SMG ducts and acini in dl SMGs suggests that Eda/Edar signaling is essential for lumina formation and glandular histodifferentiation. Our localization of Eda and Edar proteins at sites of lumen and acini formation supports this conclusion. Moreover, the presence of SMGs in both Ta and dl mutant mice, as well as the absence of immunodetectable Eda and Edar protein in Initial Bud and Early Pseudoglandular stage SMGs, indicate that Eda/Edar-mediated signaling is important for branching morphogenesis and histodifferentiation, but not for initial gland formation. To initially delineate the morphoregulatory role of Eda/Edar-mediated signaling during embryonic SMG development, we cultured embryonic day 14 SMGs with enhanced or abrogated Eda/Edar signaling. Eda supplementation induced a significant increase in SMG branching, and enhanced activation of NF-kappaB. Abrogating Eda/Edar signaling by adding the soluble form of Edar to bind endogenous ligand in embryonic SMGs results in a significant dose-dependent decrease in branching morphogenesis. Taken together, our results suggest that the Eda/Edar/NF-kappaB pathway exerts its effect on SMG epithelial cell proliferation, lumina formation, and histodifferentiation., (Copyright 2003 Wiley-Liss, Inc.)

The aquaporin (AQP) family of membrane channel proteins function as selective pores through which water, glycerol, and other small solutes cross the cell plasma membrane. To date, 11 members of this transporter family, designated AQP0-10, have been cloned and characterized in humans. The AQPs are differentially expressed in temporospatial patterns, where different AQPs demonstrate distinct tissue distributions that may reflect differing cell membrane transport functions. The purpose of this study was to evaluate AQP expression in the developing human teeth by RT-PCR and Western blot analysis. To access the generality of AQP expression, selected other orofacial tissues were studied by RT-PCR. The presence of all eleven human AQPs was screened in each tissue by RT-PCR. Positive amplification products were verified by direct DNA sequencing. AQPs 1, 3, 4, 5, 6, and 10 were identified by RT-PCR in developing teeth, and AQP1, 3, 5, and 6 were confirmed by Western blot analysis. AQP 4 was not detected by Western blot analysis, and we were unable to test for the recently identified AQP10 due to unavailability of antibodies. AQPs detected in other orofacial tissues by RT-PCR included gingiva (AQP3, 7, 10); Meckel's cartilage (AQP1, 3, 4, 5, 6); submandibular gland (AQP1, 3, 4, 5, 6, 7); masseter muscle (AQP1, 3, 4, 7, 8, 9,10); and infrahyoid muscle (AQP1, 3, 4,10). These results demonstrate that multiple aquaporins are expressed in developing teeth and in selected orofacial tissues.

Homeobox-containing (Hox) genes play important roles in development, particularly in the development of neurons and sensory organs, and in specification of body plan. The Hmx gene family is a new class of homeobox-containing genes defined by a conserved homeobox region and a characteristic pattern of expression in the central nervous system that is more rostral than that of the Hox genes. To date, three closely related members of the Hmx family, Hmx1, Hmx2, and Hmx3, have been described. All three Hmx genes are expressed in the craniofacial region of developing embryos. Here we show, for the first time, the expression of the transcription factor Hmx3 in postnatally developing salivary glands. Hmx3 protein is expressed in a cell type-specific manner in rat salivary glands. Hmx3 is present in both the nuclei and cytoplasm of specific groups of duct cells of the submandibular, parotid, and sublingual glands. Hmx3 expression increases during postnatal development of the submandibular gland. The duct cells show increasing concentrations of Hmx3 protein with progressive development of the submandibular gland. In contrast, the acinar cells of the three salivary glands do not exhibit detectable levels of Hmx3 protein.

Carbohydrate metabolism was examined in the developing rat salivary glands by analysing enzymatic activity and glycogen content in the postnatal parotid and submandibular glands. The following enzymes of the carbohydrate metabolism, hexokinase (HK), phosphofructokinase-1 (PFK-1), pyruvate kinase (PK), glucose-6-phosphate dehydrogenase (G6PD), and lactate dehydrogenase (LDH) as well as the content of glycogen were determined in the salivary glands of rats aged 2, 7, 14, 21, 30 and 60 days. The specific activity of HK increased from days 2 to 21 and then it decreased up to 60 days old. The values found for the submandibular glands were from 2.5 to 4.9 times higher than those found for the parotid gland, except for rats aged 60 days. PFK-1 showed a different pattern of variation between the glands. In the submandibular gland there was a statistically significant increase in PFK-1 specific activity from 2 to 30 days of age and then, in the 60 days old group a return to level of the rats aged 2 days. In parotid gland, the specific activity of PFK-1 decreased between 2 and 7 days of age, from 7 to 14 days the specific activity increased markedly and from 14 to 60 days old it gradually decreased. The specific activity of PK followed the same pattern of variation in the submandibular and parotid glands, showing no great variation. The specific activity of LDH decreased from 2 to 60 days old in the submandibular glands. In the parotid glands the mean values for this enzyme were higher for the 2 days old group, and then decreased to remained more or less constant. The potential capacity of the pentose phosphate pathway was greater than that of glycolysis at early ages. The glycogen content showed similar variation in both glands. It was initially high and then decreased. In conclusion, our results on the activities of enzymes involved in carbohydrate metabolism in submandibular and parotid glands may be relevant to the initiation of saliva secretion in these animals.

Wild rodents (Bolomys lasiurus) of both sexes were caught in a cerrado grassland area during the dry (July-September) and rainy (January-March) seasons of Brazil. Fasted animals were perfused with Karnovsky fixative through the left ventricle, under ether anesthesia, and the submandibular gland was processed for embedding in historesin. Histological and histometric data show sexual dimorphism at both seasons. In the volume percentage of the granular convoluted tubules (GCT) and their secretory granules, the males exhibited higher values. The absolute volume occupied by these structures, however, was dimorphic only in the rainy season. The diameter of the GCT, the height of its epithelium, and its total length were also greater in males during the rainy season. The absolute volumes of the acini and of the ductal tree were identical in both sexes in the dry and rainy seasons but the acinar diameter increased in the males and females during the rainy season. The sexual dimorphism and the seasonal variations now described in the B. lasiurus submandibular glands could be explained by the augmented reproductive activity of the males in the rainy period., (Copyright 2002 Wiley-Liss, Inc.)

Developmental changes (from 2 to 26 weeks) in phosphodiesterase (PDE) activity in the rat submandibular gland were investigated. Major activities for both cAMP- and cGMP-PDE were present in the 100000 x g supernatant fractions (70-90% of total activities), but not in the pellet fractions, during development. The effects of stimulators (Ca(2+)/calmodulin and cGMP) and inhibitors (cGMP, cilostamide, rolipram and zaprinast) were investigated in the supernatant fractions. During development, PDE4 (cAMP-specific PDE) was a major PDE, indicating that the majority of cAMP is hydrolysed by PDE4. In the young rat, PDE1 hydrolysed cGMP three-fold more than the control, and PDE2 (cGMP-stimulated PDE) was present, indicating that the concentration of intracellular cGMP may be enhanced, and cGMP may function in the growth pathway in the submandibular gland. Chromatograms eluted on a Mono Q HR5/5 ion-exchange column supported the results of the inhibition studies: PDE1, PDE2, PDE3, PDE4 and PDE5 were present in the young submandibular gland, and PDE1, PDE3, PDE4 and PDE5 in the adult gland. Expression of PDE5 was detected by inhibition studies, reverse transcriptase-polymerase chain reaction and Western blotting in the submandibular gland.

The granular convoluted tubule (GCT) cells of the submandibular glands represent a major production site for epidermal growth factor (EGF). This study investigates EGF production in the submandibular glands in relation to beta-adrenergic stimulation. Rats were treated with isoproterenol (beta-agonist), which caused up to a 400% increase in submandibular tissue weight after 3 weeks. The weight increase coincided with marked morphologic changes, with degranulation and an apparent decrement in the number of the GCT cells. Immunostaining against EGF revealed a reduction in the number of EGF-immunoreactive cells. Concomitantly, the glandular contents of 6-kDa EGF decreased from 12.86+/-3.42 nmol/gland (mean+/-S.E.M.) in controls to 0.26+/-0.03 nmol/gland. EGF mRNA levels, expressed relative to total RNA levels, only tended to be reduced after 3 weeks as judged from RT-PCR and in situ hybridization (ISH). The isoproterenol-treated rats had increased output of EGF in the saliva, but the salivary secretion of protein was also increased. In both glandular tissue and saliva, gel filtration revealed partially processed high molecular weight forms of EGF in the isoproterenol-treated rats. These data indicate that isoproterenol treatment leads to a hyperstimulatory state of the GCT cells, which then causes depletion of the cellular stores of mature EGF, and most likely due to a shortened posttranslational transit, incomplete peptide processing.

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.

Our laboratory has generated a genetically mutant mouse in which the alpha subunit of the heterotrimeric GTP binding protein, G(z) has been made dysfunctional by homologous recombination to determine its in vivo function. These animals show a characteristic failure to thrive phenotype. G(z alpha) is expressed in a variety of nervous system tissues as well as in the adrenal medulla. We therefore examined the autonomic nervous system of the G(z alpha) deficient mouse by measuring the activity of tyrosine hydroxylase and choline acetyltransferase in the superior cervical ganglia, submaxillary gland and the adrenal medulla. Preliminary results using animals of mixed BALB/c and C57BL/6 strains gave inconsistent results. Further experiments demonstrated differences in the activity of tyrosine hydroxylase and choline acetyltransferase between BALB/c and C57BL/6 mouse strains. The analysis of the pure strains showed a reduction in the size and enzyme levels of the adrenal gland and submaxillary glands of the G(z alpha) deficient mouse suggesting a role for adrenal insufficiency and/or nutritional disorders for the failure to thrive phenotype. The survival of sympathetic and sensory neurons was also examined in the G(z alpha) deficient mouse and in the presence of pertussis toxin, sympathetic but not sensory neuronal survival in G(z alpha) deficient mice was significantly attenuated. This suggests that in vivo other pertussis toxin sensitive G proteins may be recruited to compensate for the loss of G(z alpha).

Acidic beta-galactosidase (beta-gal) is expressed at high concentrations in the salivary glands, where its function is unknown but probably related to lysosomal catabolism of glycolipids or glycoproteins such as mucin. Here, the developmental pattern of the enzyme activity in the submandibular gland of B10A/SGSNJ mice was investigated in relation to the expression of mucin. Biochemical studies indicated that the activity of acidic beta-gal increases significantly from birth to 3 weeks of age (P<0.05). Histochemical staining of the submandibular glands of embryonic day 15 (E15), E18, 3-week-old and adult mice showed a trend of increasing staining intensity accompanied by a change in the cellular distribution of enzyme activity from E15 to adulthood. Acidic beta-gal activity was first evident at E18, around the time mucin production starts, primarily in terminal-bud epithelial cells. The spatial and temporal coordination between the appearance of beta-gal and embryonic mucin in the terminal buds of acinar cells might be related to a role of this enzyme in lysosomal degradation of embryonic mucin. In contrast, in adult mice, beta-gal activity was primarily found in granular-duct cells. The function of beta-gal in duct cells is unclear, but the change in the cellular distribution of beta-gal in the mouse submandibular gland could reflect a change in the substrate specificity of this enzyme between E18 and adulthood.

It has been suggested that the 27 kDa heat-shock protein (Hsp27) plays a role at crucial cellular checkpoints for proliferation, apoptosis, and differentiation. We examined the immunolocalization of Hsp27 in the rat submandibular gland during postnatal development, wherein acinar cells proliferate and differentiate at earlier postnatal periods. At 2 weeks of age, weak Hsp27 immunoreactivity was distributed diffusely over all gland components. At 3 weeks, Hsp27 immunoreactivity disappeared in most parts of the acini and ducts, but was intensely accumulated in a small cell population located in the acinar center. This population was composed mostly of terminal tubule (TT) type I cells. At 4 weeks, the Hsp27-immunopositive cell population in the acinar center was composed primarily of immature (type II) acinar cells, partly of immature (granulated) intercalated duct (ID) cells, and occasionally of apoptotic cells. After 5 weeks, all acinar components became mature and were no longer immunoreactive for Hsp27. When acinar cell differentiation was accelerated by administration of isoproterenol to 3-week-old rats for 7 days, the number of Hsp27-positive cells was significantly lower than in the control gland at 4 weeks, confirming that Hsp27 expression is downregulated in mature acinar cells. These results suggest that at around 3-4 weeks in postnatal development, the centroacinar TT cells stop proliferating and begin to differentiate into acinar and ID cells, and occasionally undergo apoptosis. Hsp27 is transiently expressed in the centroacinar TT cells during this critical period, and thus may play a role in their differentiation into the immediate descendants., (Copyright 2001 Wiley-Liss, Inc.)

Direct and indirect staining procedures were developed to characterize sialoglycoconjugates in developing rat submandibular gland. Lectin histochemistry, with and without prior sialidase digestion, combined with differential oxidation and deacetylation procedures was performed in situ. This allowed the expression of sialic acids to be followed during acinar cell development. It was found that terminal periodate-labile sialic acids linked to beta-galactose occurred early. In contrast, the terminal disaccharide sialic acid-N-acetylgalactosamine was only detectable at the adult stage and so was considered to be a good marker of the full maturity of this gland. The developing acinar cells were mainly characterized by C4-acetylated sialic acids belonging to short side-chains. Dimorphic expression of sialoglycoconjugate components was evident by postnatal day 44.

The postnatal development of granular convoluted tubules (GCT) in the duct system of the rodent submandibular gland is known to be androgen-dependent, but the underlying molecular mechanism is unclear. To test the possible role of the transcription factor, cyclic AMP response element-binding protein (CREB), in the androgen-induced differentiation of GCT, the effect of testosterone on the expression and localization of epidermal growth factor (EGF), a marker of GCT cells, and of CREB was examined in the submandibular glands of immature 3-week-old rats. Northern blotting demonstrated increases in both EGF and CREB mRNA 1-4 days after testosterone administration. Immunoprecipitation also indicated that CREB protein was increased in amount with testosterone administration, and that induced CREB was phosphorylated at the serine residue as in the active form of CREB. In situ hybridization demonstrated that cells with CREB mRNA signal first appeared in the distal portions of striated ducts at 1 day and had increased in number by 4 days after giving testosterone, when cells with EGF mRNA signal became evident in the same duct portions. Immunohistochemistry also showed the occurrence of CREB protein in the nuclei of duct epithelial cells before their differentiation into EGF-positive GCT cells. Finally, pieces of submandibular gland from immature rats were cultured in vitro and their expression of EGF mRNA analysed by the reverse transcriptase-polymerase chain reaction. Testosterone in the medium caused a marked enhancement of EGF expression in the gland in 1-4 days, which was attenuated by simultaneous administration of the antisense oligonucleotide for CREB as well as that for the androgen receptor. These results suggest the CREB is upregulated by androgen and has a crucial role in androgen-induced differentiation of GCT in the duct system of the rat submandibular gland.

We previously established a rat submandibular gland (SMG)-derived epithelial cell line (RSMG-1) to study the mechanism of morphogenesis in salivary gland development and regeneration. We found that activin A regulated the branching morphogenesis of RSMG-1 cells, suggesting that it is involved in SMG morphogenesis. We used a subtraction cloning procedure with activin-A-treated and untreated RSMG-1 cells to identify activin-A-induced genes. One of the genes detected encoded a rat homologue of Sel-1l (rSel-1l). rSel-1l is a mammalian homologue of C. elegans sel-1, which is a negative regulator of Notch signaling. In this study, we confirmed that activin A induces rSel-1l mRNA expression in RSMG-1 cells, and that rSel-1l is expressed in SMG acinar cells. These results suggest that activin A regulates the differentiation of RSMG-1 cells to acinar cells., (Copyright 2001 Academic Press.)

Objectives: Salivary gland organogenesis was evaluated in NOD mice, an animal model for autoimmune exocrinopathy, to determine when disease onset is first present in the target tissues., Methods: Submandibular glands were removed for histological, immunohistochemical and biochemical evaluation from neonatal NOD and congenic strains as well as healthy control C57BL/6 mice., Results: Histomorphological analyses of neonatal submandibular glands, the primary target for autoimmune exocrinopathy at 1 day postpartum, revealed delayed morphological differentiation during organogenesis in autoimmune-susceptible NOD mice when compared to nonsusceptible C57BL/6 mice. Acinar cell proliferation was reduced, while expression of Fas, FasL and bcl-2 were increased. Acinar cell proliferation was reduced, while expression, of Fas, FasL and bcl-2 were increased. Throughout the preweaning period (21 days) submandibular glands from NOD and NOD congenic strains aberrantly expressed an increased matrix metalloproteinase (MMP)-2 and MMP-9 activity. Substitution of two susceptibility alleles (Idd3 and Idd5) in NOD mice resulted in an hierarchical and additive reversal of delayed organogenesis, elevated MMP-9 activity, and aberrant expression of parotid secretory protein., Discussion: NOD-derived mice whose submandibular glands showed normal organogenesis did not progress to develop autoimmune exocrinopathy. Altered organogenesis of target tissue may therefore provide a cellular microenvironment capable of activating autoimmunity., (Copyright 2001 S. Karger AG, Basel)

We studied the involvement of the apoptotic mechanism(s) in cell differentiation in the developing male rat submandibular gland using the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-labeling) assay in combination with light and electron microscopy. Whereas the proacinar cells were completely transformed into acinar cells within 2 weeks after birth, starting on postnatal Day 21, the terminal tubule cells formed vacuoles that disappeared by postnatal Day 35. During this period, positive TUNEL reactivity was seen in the terminal tubule cells, and electron microscopic analysis showed that certain morphological features of apoptosis, including fragmentation of nuclei and the presence of apoptotic bodies in the cytoplasm, were present in and restricted to the terminal tubule cells. These results indicate that, in addition to an autophagocytosis-mediated mechanism, apoptosis may also be involved in reducing the number of terminal tubule cells during postnatal development in the submandibular gland.

Programmed cell death, or apoptosis, occurs during the development of many tissues and organs in almost all multicellular organisms. Although apoptosis of salivary gland cells has been demonstrated in several pathological conditions, the role of apoptosis in the postnatal development of the salivary glands is unknown. We have studied the development of the rat submandibular gland (SMG) during its transition from the perinatal stage to the mature adult stage. Terminal tubule or Type I cells, which synthesize the secretory protein SMG-C, are prominent in the perinatal acini and are believed to form the intercalated ducts of the adult gland. Between 25 days and 30 days after birth, the number of Type I cells and their SMG-C immunoreactivity markedly decreased. Apoptotic cells in association with the developing intercalated ducts were labeled with the Terminal Deoxyribonucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) method. Between 25 and 40 days of age, from 50 to 80% of the apoptotic cells in cryostat sections of the SMG were closely associated with the intercalated ducts. Electron microscopy showed that the Type I cells became vacuolated, their secretory granules were reduced in size and number, and the amount of rough endoplasmic reticulum was decreased. Cellular debris resembling apoptotic bodies was phagocytosed by macrophages and adjacent intercalated duct cells. These observations suggest that the loss of Type I cells and reduction of SMG-C immunoreactivity during development of the intercalated ducts of the adult rat SMG is due, at least in part, to apoptosis., (Copyright 2000 Wiley-Liss, Inc.)

Carbonic anhydrase has been localized to the acini and ducts of mature rat salivary glands. This enzyme has been associated with ion transport, a prominent function of striated and excretory ducts in salivary glands, suggesting that it might be used as a marker of ductal differentiation. The purpose of this study was to immunohistochemically document developmental changes in carbonic anhydrase in the ducts of the rat sublingual and submandibular glands. Immunohistochemistry was performed with antibodies to human carbonic anhydrase isoenzymes I, II and VI on sections of sublingual and submandibular glands from rats at representative postnatal developmental ages. Reactions were weak in the ducts of both glands at 1 day, then progressively increased. By 42 days, reactions had the adult pattern of virtually none in the mucous or seromucous acini, moderate to strong in the striated and excretory ducts, and none to weak in the intercalated ducts. Weak to moderate reactions were observed in the granular convoluted tubules of the submandibular gland as they became recognizable at age 42 days. Reactions to carbonic anhydrase I and II antibodies also increased from none (1 day) to modest (42 days) in the demilunes of the sublingual gland. The order of reaction intensity of the antibodies was II > I > VI. When localized via these anti-human antibodies, carbonic anhydrase is a useful marker of the functional differentiation of the striated and excretory ducts of the developing rat sublingual and submandibular glands., (Copyright 2000 Wiley-Liss, Inc.)

The expression of the gap-junction proteins connexin 32 and 43 in the developing rat submandibular gland was determined using reverse transcription-polymerase chain reaction and immunohistochemistry. Sprague-Dawley rats from the 17th gestational day through to the 28th postnatal day were used. Connexin 43 gene expression was detected on the 17th gestational day. Connexin 32 gene expression was also detected on the 17th gestational day but less intensely. Immunolabelling for connexin 43 was also found in the developing submandibular gland from the 17th gestational day. Prenatally the most immunoreactive areas for connexin 43 were located in the periphery of terminal tubules, but some staining was discernible between the cells in terminal buds. In the postnatal period, reactive areas were located at both the periphery of acinus-like structures and around the intercalated duct. This is consistent with the known association of connexin 43 with myoepithelial cells. Connexin 32 immunostaining was first detected in the developing submandibular glands on the 18th day of prenatal development. Positive staining was present on the lateral side of the proacinar and mature acinar cells. The number of immunoreactive areas per cell increased during early development followed by a significant decrease perinatally. During postnatal development the density of areas again showed a pattern of increase. These results suggest that connexin 43 is associated with growth and differentiation in the pre- and perinatal periods and also with the contractile function of myoepithelial cells in the postnatal period of the developing submandibular gland. It is also implied that the number of connexin 32-positive areas may correspond to an increase or decrease in the number of proacinar and mature acinar cells.

We studied the distributions of laminin-5 and hemidesmosome components, HD1/plectin and BP230, in the submandibular glands of adult and developing mice. In adult mice, laminin-5 was expressed in the basement membranes of both the myoepithelial cells and excretory ducts. The former expression was predictable because laminin-5 is a ligand for hemidesmosomes, which appear in myoepithelial cells and stratified epithelium. However, the latter expression pattern suggested that the non-stratified epithelium of the excretory duct might also be associated with hemidesmosomes. During fetal development, laminin-5 was found in the basement membrane of developing ducts but not epithelial end buds in which future lobules are formed by epithelial branching. The expression of HD1/plectin but not BP230 was noted in the developing duct at early embryonic stages, indicating the presence of type II hemidesmosomes. Expression of BP230 appeared in the excretory duct epithelium at around the day of birth. At this stage, the typical hemidesmosome was observed in the duct epithelium. Our results suggest that laminin-5 is involved in duct development rather than epithelial branching. The results also suggest that the developing duct epithelium interacts with laminin-5 through the type II hemidesmosome, which later matures into a typical hemidesmosome upon the onset of expression of BP230.

Anti-laminin serum was used to investigate distribution patterns and chain composition of laminin during postnatal development of rat submandibular gland. Immunohistochemical analysis showed that in glands of newborn rats laminin was not uniformly present around growing acini. Staining was frequently weak or absent in clefts formed between adjacent cells. This irregular staining pattern decreased progressively over the subsequent periods, and in 30-day-old animals immunoreactivity was observed only at the periphery of glands. Immunoblot analysis showed that laminin was composed of bands corresponding to the alpha 1, beta 1 and gamma 1 polypeptides. The correlation between the pattern of laminin expression and gland maturation suggests a role of laminin in the functional maturation of acinar cells.

Although the submandibular gland (SMG) plays important exocrine and endocrine roles, little is known about the molecular details underlying its development. Previously, we reported that in the postnatally developing hamster SMG, GPT, the protein product of the first N-glycosylation gene, ALG7, was an in vivo marker for salivary cell proliferation. Here we investigated the proliferative, cytoskeletal, and adhesive changes during SMG postnatal development. The cellular localization and abundance of GPT, filamentous actin, and beta1 integrin receptor were examined using confocal microscopy and immunoblotting. In neonatal glands, high GPT levels marked extensive cell proliferation throughout the tissue. The apical regions of immature salivary cells displayed intense actin staining, while most of the beta1 integrin was diffusely distributed throughout the tissue. As development proceeded, discrete regions of the gland expressed attenuated levels of GPT, an increased organization of actin to the cell cortex, and beta1 integrin to the basal lamina. In the adult SMG, differentiated salivary cells displayed low levels of GPT and actin. While the abundance of beta1 integrin remained unchanged throughout development, in the adult, it was found exclusively in regions where cells contact the basal lamina. These data indicate that SMG development entails regionalized cell proliferation and polarization, and that these processes are temporally and spatially coordinated with the establishment of stable cell-substratum interactions.

The rat submandibular gland is not fully developed at birth and definitive differentiation takes place postnatally. The steady-state mRNA expression for the four proteinase inhibitor molecules, tissue inhibitors of metalloproteinase (TIMP)-1 and -2, and cystatins S and C, and for a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), in rat submandibular glands was measured by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) at different stages of postnatal development. The gene-expression patterns of TIMP-1 and -2 relative to G3PDH were similar to each other. The TIMP-2 and cystatin C genes were more highly expressed than those of TIMP-1 and cystatin S at all stages. Moreover, the gene expressions of TIMP-1 and -2, and of cystatins S and C, were predominant between 1 and 7, and 7 and 12 weeks of age, respectively, and coincided developmentally with the regression of terminal tubule cells and the differentiation of granular convoluted tubule cells, respectively. Quantitative competitive RT-PCR allowed accurate measurement of small changes in the steady-state concentrations of these proteinase-inhibitor mRNA molecules.

S100 proteins are calcium-binding proteins of the EF-hand superfamily and are involved in the regulation of a number of cellular processes. The present study deals with the immunohistochemical expression of S100A1 and S10100A6 in the rat submandibular and sublingual glands during postnatal development from day 0 to 12 weeks. Expression of S100A1 was particularly concentrated in pillar and transition cells in the granular convoluted tubule (GCT) and in striated duct cells of the submandibular gland age 4 weeks to maturity. None or only weak staining for S100A1 was observed in the duct segment at 0-5 days. On the contrary, immunostaining of S100A6 was present in proacinar cells in undifferentiated submandibular gland at age 3 days to 2 weeks. S100A6 immunoreactivity in rat submandibular gland coexisted with chromogranin reactivity. It is possible that S100A6 regulates secretion of chromogranin in proacinar cells. Secretion of growth factors and biologically active peptides in the GCT are regulated by calcium signals, and S100A1 may be involved in the secretory mechanism of granular cells. S100A1 and S100A6 are potentially of great importance in secretory functions of granular cells and proacinar cells, as well as in rat salivary glands.

Tuft cells, a widespread cell type that is present in the mucosal epithelia of hollow organs, including the main excretory duct (MED) epithelia of the rat salivary gland, are well documented morphologically. However, studies of their development are few. The purpose of the present study was to examine the perinatal and postnatal development of tuft cells in the main excretory duct of the rat submandibular gland. Main excretory ducts of the submandibular gland were obtained from five male Wistar rats at the ages of 0, 1, 7, 14, 17, 21, 23, 28, and 56 postnatal days and were prepared for scanning and transmission electron microscopy. The tuft cells, which are distinguished easily by their long microvilli protruding into the lumen, were recognizable first at 17 postnatal days. They showed a remarkable increase in number between 3 and 4 postnatal weeks. The percentages of tuft cells were 0.4% at 17 postnatal days and 0.8% at 3 postnatal weeks. The number of tuft cells represented approximately 5% of the total epithelial cells by 4 postnatal weeks. There was a significant difference between 3 and 4 postnatal weeks (P < 0.01). The microvilli of the tuft cells at the time of weaning had almost the same width as in the adult, but they were shorter. Microfilaments extending from the tips of the microvilli and microtubules and many electron-lucent vesicles in the supranuclear cytoplasm also were observed. These results indicate that tuft cells appeared in the MED of the submandibular gland during weaning and had abundant vesicles in their apical cytoplasm.

Cyclic AMP response element-binding protein (CREB) is a 43-kDa polypeptide that binds a cAMP response element located at the 5' promoter region of cAMP regulatory genes. The spatial and temporal distribution of CREB in the postnatal development of the rat submandibular gland was investigated using immunohistochemistry with a specific antibody. At birth, cells of the terminal tubules and ducts in the submandibular gland showed a nuclear CREB immunoreactivity of moderate intensity. At 1-2 weeks after birth, an intense CREB immunoreactivity was localized primarily to acinar cells. When the beta-adrenergic agonist isoproterenol was administered to 2-week-old rats, a twofold transient increase in the number of immunoreactive acinar cells was induced. Beginning 3 weeks after birth, CREB immunoreactivity shifted from acini to the duct system and showed a clear localization in the cells of the intercalated ducts and distal portions of striated ducts, where the granular convoluted tubule develops after 4 weeks. Immunopositive materials were localized exclusively in the nuclei of both acinar and ductal immunoreactive cells. After the development of the granular convoluted tubules, CREB immunoreactivity was absent in the tubule cells and was gradually reduced in intensity over the entire gland. In order to examine a hypothesis that CREB is involved in the initial differentiation of the granular convoluted tubular cells, testosterone was administered to hypophysectomized adult rats. Whereas the tubular cells of hypophysectomized rats showed a complete regression, and no CREB immunoreactivity was found in any acinar or duct cells, administration of testosterone for a few days induced an intense CREB immunoreactivity in the nuclei of duct cells, followed by their differentiation into the granular convoluted tubular cells. These results suggested that CREB is involved not only in the growth and differentiation of acinar cells that are regulated by beta-adrenergic nerves but also in those of the duct system, and especially in the androgen-regulated differentiation of the granular convoluted tubular cells, during the post-natal development of the rat submandibular gland.

In addition to seromucous cells and a few mucous cells (Type II cells), submandibular glands of 2-8 month old rats contain two additional cell types: type III and type IV. Type III cells contain serous-type secretory granules that sometimes have a complex substructure; type IV cells appear to be seromucous, but their granules clearly are different from those in conventional endpiece seromucous cells. Both type III and IV cells are involved in histogenesis of new endpieces in a process that differs markedly from that occurring in perinatal glands. In this process, intercalated ducts bud and give rise to immature endpieces that consist entirely of type III cells. These differentiate into type IV cells, which in turn differentiate into standard seromucous cells. Concurrently, the intercalated ducts become shorter as their most distal cells differentiate into granular duct cells. This type of developmental process begins approximately 2 months postnatally, when histogenesis of endpieces by means of terminal tubules has ended, and continues until 6 months, when its frequency sharply declines.

Secreted carbonic anhydrase (isozyme VI; CA VI) was localized by immunohistochemistry in the developing postnatal rat submandibular and parotid glands using a specific monoclonal antibody to the rat enzyme. CA VI immunostaining was not detectable in the glands before birth. In the submandibular gland, granular immunostaining for CA VI was detectable in several terminal tubule cells of 1-day-old rats. At 1 week, the CA VI-positive cells were located at the periphery of the terminal tubules and appeared to be budding off the tubules. These cellular buds gradually increased, and, by 4 weeks, formed acini. CA VI was also detected in the duct lumen from day 1. The immunostaining in the parotid gland was detected sporadically in the acinar cells at 2 or 3 weeks. By 4 weeks, when the gland was almost indistinguishable from the adult one, the number of positive acinar cells had increased. Their number, however, was far smaller than in the adult gland, and the enzyme could not be detected in the duct lumen. CA II was also localized using specific antibodies to the rat isozyme. CA II was detectable in the inter- and intralobular striated ducts at 2 weeks after birth in the submandibular gland and at 3 weeks in the parotid gland. These results suggest that CA VI is secreted into saliva from soon after birth and that CA II appears in parallel with the functional maturation of the ducts. In addition, CA II was transiently expressed by the cellular buds of the submandibular gland at 2 and 3 weeks.

Lung epithelium during morphogenesis maintains a sheet structure of polarized cells lining a lumen, in which E-cadherin, beta-catenin and tight junctional proteins are localized at the cell-cell contact sites. On the other hand, the submandibular gland epithelium at early stages of development forms a non-cavitated mass of cells where E-cadherin/beta-catenin are present on the entire cell surfaces and tight junctional proteins are almost absent or weakly scattered. In the present study, tissue recombination experiments were performed between the two organs to explore roles of mesenchyme in the architectural development of the epithelium. Homotypic recombinants of both submandibular gland and lung showed the tissue architecture as observed in the intact organs. In contrast, 11-day lung epithelium cultured with 13-day submandibular mesenchyme formed multilayers of cells with the lumen being less visible. It was accompanied by redistribution of E-cadherin/beta-catenin along the entire cell surfaces and by an irregular distribution of tight junctional proteins. A similar redistribution of these molecules was observed in 15-day lung epithelium cultured with the submandibular mesenchyme, although the epithelial sheet structure lining the lumen was formed. On the other hand, the tissue architecture of submandibular gland epithelium was little affected by lung mesenchyme, although the epithelium was flattened and showed branching morphogenesis.

Mouse submandibular salivary gland (SMG) mucin is the primary histodifferentiation product of submandibular epithelia. We demonstrate marked differences between embryonic, neonatal, and adult SMG mucin mRNA and protein by Northern and Western blot analyses: E17 and 1-day-old neonates exhibit two unique mucin transcripts (1.20 and 0.85 kb) which are approximately 19% greater or smaller in size than the single (1.01 kb) adult transcript. Two embryonic protein isoforms (Mr approximately 110 and 152 kDa) are immunodetected compared to a single adult protein (Mr approximately 136 kDa), with the larger (approximately 152 kDa) embryonic isoform persisting in neonatal glands. Mucin transcripts are localized to the branching epithelia in E14 and older SMGs, with increased hybridization signal being seen in terminal bud and proacinar epithelial cells with age; a significant 26% increase in transcript levels is detected by RNase protection assay between E14 and E19. By contrast, submandibular mucin protein is not immunodetected until E17, being primarily immunolocalized to terminal bud and proacinar epithelial cell membranes. Our data clearly shows that substantial qualitative differences exist between embryonic and adult SMG mucin mRNA and protein., (Copyright 1998 Elsevier Science Ireland Ltd. All rights reserved)

The data presented here provide evidence that the abundance of the ALG7 protein product, GPT, correlates with high proliferative activity during the postnatal development of the hamster SMG development, and that it becomes downregulated with differentiation. Based on our previous studies with yeast, changes in the level of ALG7 expression may be necessary for the events directing salivary cell polarization, migration, differentiation, and apoptosis at distinct developmental stages.

Experiments were performed to document the presence of G protein-coupled P2Y nucleotide receptors in rat salivary glands and to examine changes in receptor expression during development and under conditions in which gland architecture is altered. The results indicate that, as opposed to mature rat submandibular gland (SMG), immature glands express functional P2Y1 receptors. P2Y1 receptor activity was highest at birth and declined over the next four weeks to undetectable levels. P2Y1 receptor mRNA levels remained constant over this time course, suggesting that receptor activity is regulated at some point other than transcription. Conversely, short-term culture of cells from the three major salivary glands resulted in upregulation of functional P2Y2 receptors. Responses to the P2Y2-selective agonist, UTP, were obtained after 3 h in culture and were maximal by 72 hours. This increase was paralleled by increased steady-state P2Y2 receptor mRNA levels. Upregulation of P2Y2 receptors also occurred in vivo following ligation of the main excretory duct of the SMG. These studies suggest that nucleotide receptors are dynamically regulated during development and as a result of perturbations to gland architecture.

The concentrations of principal neurotransmitters in the submandibular, parotid and sublingual glands were compared between two pairs of age-matched male and female ddY mice, one pair consisting of 4-week-old and the other 8-week-old animals. Sex-dependent differences in both noradrenaline and acetylcholine concentrations were observed only in the submandibular gland, although each neurotransmitter showed distinct features. The acetylcholine concentration in the submandibular gland was higher in the female at both ages, whereas the noradrenaline concentration was higher in the female at the age of 4 weeks but became higher in the male by the age of 8 weeks. On the other hand, the total amounts of noradrenaline and acetylcholine per submandibular gland were already greater in the male at 4 weeks, and the male parotid and sublingual glands also had a greater noradrenaline content by 4 weeks and 8 weeks, respectively. Each type of gland had similar growth rates over the 4-week period, and the male submandibular and parotid glands were heavier than the female. In addition, each type of gland had its characteristic ratio of noradrenaline to acetylcholine concentration, which did not differ between the sexes and remained in similar basic patterns during the period examined, except for the submandibular gland of 8-week-old male mice, which developed greater amounts of the sympathetic neurotransmitter noradrenaline.

The rat submandibular gland is innervated by both sympathetic and parasympathetic branches of the autonomic nervous system which, in turn, regulate the secretory function of the gland. Parasympathetic innervation of rat submandibular glands is present at birth; in contrast, sympathetic innervation reaches the glands by postnatal day 5. Isoproterenol (IPR), a beta-adrenoreceptor agonist, induces hypertrophic and hyperplastic enlargement of rat salivary glands, and induces the expression of a number of genes, including cystatin S (Cys S), a member of family 2 of the cysteine proteinase inhibitor superfamily. Cys S gene expression is tissue specific, cell type specific, occurs temporally during normal development and is not observed in adult animals unless stimulated by IPR. In addition, sympathectomy of adult rat submandibular glands reduced IPR-induced expression of the Cys S gene. This paper reports experiments analyzing the participation of the sympathetic branch of the autonomic nervous system in IPR-induced expression of the cystatin S gene during early development of the submandibular gland. The rat Cys S gene could be induced by IPR by day 3, and the level of Cys S mRNA remained constant until 8 days at which time a dramatic IPR induction of Cys S mRNA was observed. This statistically significant increase in Cys S mRNA at 8 days was diminished, but not completely suppressed, upon sympathectomy of 1-day-old animals. These data indicate that an intact sympathetic innervation is not a requisite for IPR-induced Cys S gene expression in developing submandibular glands; however, sympathetic innervation is required for the full IPR response of the Cys S gene in developing submandibular glands. The developmental experiments presented in this paper indicate that factor(s) coming from the sympathetic nervous system participate in IPR-induced expression of the Cys S gene in rat submandibular glands.

Nonobese diabetic (NOD) mice develop an anti-exocrine gland pathology similar to human Sjögren syndrome. Recently, we demonstrated that NOD-scid mice develop severe loss of submandibular acinar cells with concomitant appearance of abnormal isoforms of salivary proteins suggesting de novo enzymatic cleavage. Because these changes may indicate activation of apoptotic proteases, we examined saliva and salivary tissue for cysteine protease activity. Cysteine protease activities were elevated in saliva and gland lysates from 20-week-old NOD and NOD-scid mice as compared with age- and sex-matched BALB/c or 8-week-old NOD mice. This activity appeared in the submandibular glands, but not in the parotid glands. Western blot analyses using antibodies directed against specific apoptotic proteases (interleukin 1beta converting enzyme, Nedd-2, and Apopain/CPP 32) confirmed these findings. Submandibular glands from NOD-scid mice exhibited the greatest increase in proteolytic activity, indicating that infiltrating leukocytes are not responsible for these changes. Western blot analyses also failed to reveal changes in the levels of cystatins (saliva proteins that inhibit protease activity). Thus, increased cysteine protease activity appears to be directly related to submandibular acinar cell loss in NOD-scid mice involving the apoptotic pathway. Additional protease activity in saliva and gland lysates of older NOD and NOD-scid mice, apparently mutually distinct from cysteine proteases, generated an enzymatically cleaved parotid secretory protein. We suggest, therefore, that proteolytic enzyme activity contributes to loss of exocrine gland tolerance by generating abnormally processed protein constituents.

Carbonic anhydrase VI (CA VI) is a secreted enzyme produced predominantly by serous acinar cells of submandibular and parotid glands. We have investigated the developmental pattern of CA VI production by these glands in the sheep, from fetal life to adulthood, using immunohistochemistry. Also, a specific radioimmunoassay for CA VI was used to measure changes in enzyme expression in the parotid gland postnatally. CA VI is detectable by immunohistochemistry in parotid excretory ducts from 106 days gestation (term is 145 days), in striated ducts from 138 days and in acinar cells from 1 day postnatal. The duct cell content of CA VI declined as the acinar cell population increased, a feature also of CA VI immunoreactivity in the submandibular gland. Production of CA VI by submandibular duct cells was detectable initially at 125 days gestation, and acinar production was not seen before 29 days post-natal. Apart from the differing ontogeny of CA VI production in ducts and acini of parotid and submandibular glands, there was a parallel pattern of CA VI expression during the development of these major salivary glands. With the development of the acinar tissues in the postnatal lamb, there was a dramatic increase (about 600-fold) in the level of expression of CA VI in the parotid gland between days 7 and 59 as measured by radioimmunoassay.

The development of acinar and ductal cells of the mouse submandibular gland was studied using field emission SEM, conventional TEM and HVTEM methods. The specimens, at 15 and 18 days of gestation and 1, 3, 7, 14, 21, 30, 90 and 180 postnatal days were fixed in 2.5% glutaraldehyde solution in 0.1 M sodium phosphate buffer (pH 7.3). At 15 and 18 days of gestation, the structure of mouse submandibular gland contains acinar and ductal cells in proliferation. The cytoplasmic organelles such as mitochondria, granular endoplasmic reticulum and Golgi apparatuses are scattered in the cytoplasm. At 18 prenatal days only several acinar cells present immature secretory granules in the apical portion. In this stage the acinar and ductal cells are enveloped by bundles of fine collagen fibrils disposed in several directions. There are also numerous capillaries located closely to the acinar cell membranes. In the aging stages of 1, 3, 7, 14, 21, and 30 postnatal days, the histo-differentiation of acinar, intercalated and ductal cell components are observed. At newborn day one the cytoplasmic organelles start to place themselves around the nucleus. Several immature secretory granules are observed at day one, however, they increase in the aging days. At postnatal day 30, the cytoplasms of acinar and ductal cells are filled with a large number of secretory granules of different sizes. The stacks of granular endoplasmic reticulum and Golgi apparatus and some vesicles and free ribosomes are noted. The intercellular membranes are attached by desmosomes and cytoplasmic interdigitations. The luminal surface shows several small projections of microvilli. An electron-dense line of basement membranes followed by fine collagen fibrils are recognized. Delicate capillaries are found in the outer surface of acinar cells. At postnatal day 90 and 180 the acinar, intercalated and striated ductal cells reveal numerous secretory granules in the apical portion. The acinar cells showed basal nuclei and the parallel arrangement of granular endoplasmic reticulum. The mitochondria are located at the base of ductal cells showing a typical pattern of cristae. In these stages the intercellular digitations of cytoplasmic protrusions and desmosomes are also noted. The cytoplasm of myoepithelial cells are seen along the cell membranes. The spongy-like structures constituting the basement membrane are followed by bundles of fine collagen fibers.

The first appearance of substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), galanin (GAL), leucine-enkephalin (1-ENK), and methionine-enkephalin (m-ENK) in the male mouse submandibular glands were different for each. VIP immunoreactive fibers first appeared on embryonic day 15 (E15), SP on E16, and CGRP fibers on E18. GAL, 1-ENK, and m-ENK fibers appeared in the early postnatal period, and NPY fibers occurred on postnatal day 21 (P21). From P0 to P21, VIP fibers rapidly increased in number, but SP and CGRP fibers increased only slightly. After P21, VIP, SP, and CGRP fibers decreased in number. ENK fibers were found only from P0 to P14. The number of these immunoreactive fibers in the adult phase was low in comparison with that in early postnatal phase. Around the blood vessels, SP, VIP, CGRP, NPY, and GAL fibers appeared by at least P7. These findings suggested that the transient high activity of VIP, CGRP, SP, and GAL and the transient appearance of ENKs in the nerve fibers may be related to the cell proliferation and differentiation of the functionally important structures of the mouse submandibular glands, and that the peptidergic innervation around the vasculature is probably involved in controlling local glandular circulation.

Our purpose in this work was to investigate the role of target tissues in the regulation of dendritic morphology from sympathetic neurons during development and aging. Neurons were retrogradely labeled from three targets, the iris, the submandibular gland (SMG), and the middle cerebral artery (MCA). They were then fixed and intracellularly injected to demonstrate their dendritic arborizations. Dendritic geometry varied quantitatively in sympathetic neurons innervating different target tissues at all stages of development. Neurons innervating the iris had the largest cell bodies and most extensive dendritic arborizations, whereas the vasomotor neurons were the smallest. The number of primary dendrites, however, did not vary significantly between the different neuronal populations. The growth of dendritic arborizations during postnatal development and their atrophy in old age were not concordant in the different neuron populations we studied. Neurons innervating the MCA and the iris ceased dendritic growth early in postnatal development, whereas the dendritic complexity of neurons supplying the SMG increased well into adulthood. By contrast, dendritic atrophy was seen in aged MCA- and SMG-projecting neurons but not in those innervating the iris, suggesting, with other evidence, correlated and distinct patterns of growth and atrophy in axons and dendrites of mature sympathetic neurons projecting to different targets. Swollen dendrites and protuberances on cell soma were a prominent feature of aged neurons. In addition to the target-specific variation in neuronal morphology, we observed diversity in neurotransmitter phenotype. For example, neuropeptide Y was expressed in iridial but not SMG-projecting neurons. These results show a range of age- and target-specific differences in the dendritic morphology and neuropeptide content of sympathetic neurons that may be a result of differing trophic interactions with their target tissues.

Submandibular acinar cells of 1-day-old, 7-day-old, and adult rats were analyzed with X-ray microanalysis after stimulation with carbachol for different time periods (2-7 min). In unstimulated animals, marked differences in elemental content between compartments could be observed: secretory granules had a higher Ca and lower P and K content than other cell compartments. Comparison between different age groups showed significant differences for Ca, which increased with age in all compartments; Mg increased with age in the secretory granules and the apical cytoplasm. Only the glands from adult animals showed a significant effect of cholinergic stimulation: a transient decrease in Cl and K. The Cl concentration in the secretory granules decreased to 60% of the control value, which suggests that the granules release Cl upon stimulation. In young animals, no or little change in elemental distribution was observed after stimulation. This may indicate that Cl- secretion mechanisms are much less prominent in young animals. The ultrastructure of submandibular secretory granules depends on the preparation method: condensed and electrondense in freeze-substituted unfixed tissue, decondensed and more translucent in aldehyde-fixed tissue. This may indicate that the granules can transport water, and swell during the process of aldehyde fixation.

We investigated the influence of the streptozocin (STZ)-diabetic state on the dose-response curves for salivary flow and protein content in saliva stimulated by pilocarpine and noradrenaline in mice. The diabetic state increased the relative weights of parotid and sublingual salivary glands but not the weight of submandibular glands, despite body weight loss. In the dose-response curves, (1) the maximal responses to stimulation with pilocarpine and noradrenaline on salivary flow, and with noradrenaline on protein content in saliva, were depressed by the diabetic state, and (2) the value of the 50% effective dose for salivary flow with pilocarpine, but not with noradrenaline, was decreasingly altered by diabetic mice. These results suggest that xerostomia, one of the complications of non-insulin-dependent diabetes mellitus, is caused in part by muscarinic and adrenergic receptor dysfunction in the salivary glands.

The concentration and the localization of transforming growth factor (TGF)-alpha and epidermal growth factor (EGF) in the submandibular glands (SMGs) of male Wistar rats of different ages (postnatal 0 to 10 weeks of age) were examined. Highest levels of TGF-alpha were seen early, at postnatal day 0; the levels dropped thereafter in an age-dependent manner, while EGF was not detectable before the third postnatal week. Immunoreactive localization of EGF was restricted to the granules of the granular convoluted tubule (GCT) cells in the mature SMGs, whereas TGF-alpha was observed throughout postnatal development over the entire duct system. TGF-alpha was demonstrated in the cytoplasm at early stages when the GCT granules were not observed and was also located on the granules at the late stage, as was the case for EGF, indicating that TGF-alpha is colocated with EGF in the mature SMG. These results demonstrate the differences between the expression of TGF-alpha and that of EGF in the developing rat SMG.

Previous studies have indicated that following nutrient ingestion, GIP is released principally from the upper small intestine. In addition to its presence in the rat small intestine, GIP transcripts have also been localized to the submandibular salivary gland (SSG). The present studies were directed to further characterize expression of the GIP gene in the SSG. Pregnant rats were sacrificed at gestational days 18 and 20, followed by the removal of rat fetuses. The duodenum pancreas, and SSG were then excised from the fetuses, as well as from neonatal pups at ages 1, 3, 7, 10, 14, and 21 days. RNA was extracted and measured by Northern blot analysis using specific rat GIP probes. GIP transcripts were first detected in the duodenum in the 18-day fetus and reached maximum levels at birth. In contrast, GIP mRNA was not observed in the SSG until 10 days postnatally and was not detected at all in either the fetal or neonatal pancreas. In situ hybridization of the SSG using an 35S-labelled antisense GIP RNA probe demonstrated expression of the GIP gene to be limited to ductal cells, with no transcripts present in acini. In separate experiments, rats fasted overnight were given water or 10% glucose. While no changes were detected in water-fed rats following oral glucose ingestion, small, but significant increases in SSG GIP gene expression were detected at 60 and 240 min. The results of these initial studies suggest the possibility of a functional role for GIP in the rat salivary gland by the demonstration of GIP mRNA in the SSG by Northern analysis and in situ hybridization, as well as by an increase in SSG GIP gene expression following a glucose meal.

We studied the immunohistochemical localization of androgen receptor in the mouse submandibular gland, and developmental profiles of its expression using polyclonal human androgen receptor antibody. In the submandibular glands of both sexes, specific immunoreactivity appeared only in cell nuclei of the acini, the intercalated ducts, the granular convoluted tubules (GCT) and the excretory striated ducts. The percentage of immunoreactive cells in each region gradually declined with age during the first 90 days of postnatal development studied. The sexual difference in the percentage of immunoreactive cells was observed in the acini on days 20 and 30 and in the GCT on day 30. Incidence of immunoreactive cells in the female was significantly smaller than that in the male. On day 60, the percentage of immunoreactive cells of these two regions turned to increase slightly in the female but continued to decline in the male, and then it became higher in the female than in the male. In addition, one-week castration did not cause any changes in the intracellular distribution of androgen receptor and the percentage of immunoreactive cells in each region of the adult gland. These results suggest that androgen receptor is localized primarily in cell nuclei in all four regions of the mouse submandibular gland in situ, and that its expression in acini and GCT is superior in the male around days 20 to 30, when sex difference of the gland becomes evident.

The DNA synthesis and the changes of cell population kinetic of aging male mouse submandibular glands were studied by light and electron microscopic radioautography from embryonic day 19 to 21 months postnatal. At the embryonic day 19, the gland consisted of developing acinar cells (49%), intercalated duct (ICD) (37%), juxta-acinar (JA) (3%) and striated duct (SD) cells (11%). At birth, JA cells increased rapidly to 32%, thereafter decreased gradually. At 1 month of age, granular convoluted tubule cells appeared and increased rapidly in number with age. They reached a maximum at 6 month after birth. Then they appeared to decrease gradually from 6 to 21 months. The most conspicuous feature of the aged submandibular glands was the regressive changes of the granular ducts. The extent and height of the granular ducts as well as the content of mature secretory granules decreased. These changes may play an important role during the development of mice, because the granular duct synthesizes many biological factors. The 3H-thymidine labeling index of each cell type of male mouse submandibular glands reached a peak 1 day after birth and decreased with aging. The labeling index of intercalated duct cells was higher than the others and persisted for a long time. On the other hand, the labeling indices of the other cells were significantly depressed at one week after birth. From these observations on morphology, it was also shown that ICD cells were concerned with the generation of the other cell types.

Indirect immunohistochemical methods were used to study presence and number of CGRP-immunoreactive (CGRP-IR) nerve fibers in the submandibular gland and ganglion cells of the superior cervical, submandibular and trigeminal ganglia of the developing rat. The effect of CGRP on peroxidase and total protein release was also studied in the developing postnatal submandibular glands of 1, 5, 12 and 30-day-old, as well as adult rats by in vitro methods. The possible costimulation of CGRP with SP, NKA or carbachol on 5-day-old and adult rats was also tested. The stimulatory effects of these compounds were compared to the basic release of peroxidase and total amount of proteins from submandibular gland fragments in incubation solution. CGRP-IR nerve fibers were found in relatively high number during post-natal development, mainly around blood vessels and ducts. Some CGRP-IR nerve fibers were also detected around acini. The number of these fibers was quite low and remained constant during the post-natal development. The number of CGRP-IR trigeminal ganglion cells was higher on the 5th and 12th post-natal day than later in development and in adult animals. At the same time, superior cervical- and submandibular ganglion cells were non-reactive for CGRP, suggesting trigeminal origin of CGRP-IR nerve fibers during the development in the submandibular gland. In the secretory studies, CGRP per se stimulated both peroxidase and total protein release in the submandibular gland most effectively on 5th and 12th post-natal days, while there was no clear secretory response in the adult glands. In the 5-day-old submandibular gland CGRP in combination with SP, NKA or carbachol clearly enhanced the total protein secretory response when compared with the release caused by these substances alone. However, in the adult submandibular gland, the combinations did not enhance total protein release more than any of the substances alone. Furthermore, it can be concluded that the presence of a more dense CGRP-IR innervation during the early postnatal period in the developing submandibular gland is accompanied with an increased responsiveness of the secretory elements to CGRP.

The gelatin cleaving activities in secretions of cultured fragments of male rat submandibular glands were studied using zymography. Gelatinolytic activities of 88-, 64-, and 57-kDa proteins detected in the tissues from 22-28-day old animals were undetectable in 31-70-day old rats. The traces of gelatinolytic activity associated with 28-kDa protein were detectable from 22-day old rats in serum-free media, and this activity of the enzyme markedly increased with aging from 38-days old. At 52-days and the subsequent stages, in addition to 28-kDa, activities associated with 60-, 32-, and 29-kDa proteins were strong. When the conditioned media were treated with 1,10-phenanthroline and diisopropyl fluorophosphate (DFP), both products inhibited activity of 88-kDa enzyme, indicating that this enzyme is Cls-like enzyme. The 64- and 57-kDa activities were inhibited by 1,10-phemanthroline, but not by DFP; when the conditioned medium of the tissue from 24-day old rats was treated with p-aminophenylmercuric acetate, gelatinolytic activity associated with 64-kDa converted to 57-kDa. Therefore, 64- and 57-kDa activities were concluded to be progelatinase A and gelatinase A, respectively. On the other hand, the gelatinolytic activities associated with 60-, 32-, 29- and 28-kDa proteins were inhibited by DFP but not by 1,10-phenanthroline, indicating that these enzymes belong to the family of serine proteinase, most probably kallikrein-related enzymes. From these findings, it was suggested that gelatinase A, along with Cls-like enzyme, participates in the maturation of the submandibular gland before it becomes active as an exocrine organ.

The activity of succinate dehydrogenase (SDH) was examined histochemically in the parenchyma of murine submandibular gland (SMG) during postnatal development on light-microscopical level. In this study, SDH appears as a good marker of differentiating and mature striated ducts of both sexes and convoluted granular tubules of males. Enzyme activity in glandular parenchyma increased from the 1st day to the 7th week of postnatal life. Definitive enzymatic pattern was seen in the gland aged 7 weeks. Histochemical differences between male and female SMG of mouse were noted.

The first enzyme in the dolichol pathway of protein N-glycosylation, dolichol-P-dependent N-acetylglucosamine-1-phosphate transferase, GPT, has been implicated in the development of a wide variety of eukaryotes. GPT is encoded by ALG7, an early growth-response gene, whose expression has been shown to affect the extent of N-glycosylation and secretion of proteins. To initiate the molecular characterization of ALG7 involvement in mammalian growth and differentiation, we have used the postnatally developing hamster submandibular gland (SMG) as an experimental paradigm. In this study we report that the ALG7 gene was differentially expressed during postnatal development and in terminally differentiated adult tissues. Throughout development, GPT activity paralleled ALG7 mRNA levels, suggesting that the amount of functional enzyme was determined by modulation of transcript abundance.

The expression of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) mRNA in the small intestine of suckling and adult rats was examined by in situ hybridization. EGF mRNA was found mainly in the intestinal crypts in adult rats. Adult rats also exhibited a considerably stronger signal for EGF mRNA in comparison to suckling rats, where the signal was very low or absent. In contrast to EGF, very strong expression of TGF-alpha mRNA was observed in the small intestine of both adult and suckling rats. These data suggest the differences between the expression of EGF and TGF-alpha in the developing small intestine.