Secretion by striated ducts of mammalian major salivary glands: review from an ultrastructural, functional, and evolutionary perspective.

In addition to their role in electrolyte homeostasis, striated ducts (SDs) in the major salivary glands of many mammalian species engage in secretion of organic products. This phenomenon usually is manifested as the presence of small serous-like secretory granules in the apical cytoplasm of SD cells. The composition of these granules is largely unknown, except in the case of the cat and rat submandibular gland, where the granules have unequivocally been shown to contain kallikrein. In some species, the apical cytoplasm of SD cells contains variable numbers of vesicles, both spherical and elongated, that vary in appearance from 'empty' to moderately dense. In the rat parotid gland, lucent vesicles transport glycoproteins to the luminal surface where they are incorporated into the apical plasmalemma and the glycocalyx. There is a strong possibility that in various species some of these vesicles are involved in transcytosis of antibodies to the saliva from their source (plasma cells) in the surrounding connective tissue. In addition, vesicles may engage in transfer of growth factors from the saliva to the interstitium. In a few species, conventional SDs have been replaced by ducts that are wholly given over to secretion, i.e., they entirely lack basal striations; although such ducts occupy the histological position of conventional SDs, it is not clear whether they represent a new type of duct or merely are modifications of SDs. Broad-based comparisons of ultrastructural and other data about SDs offer some insight into evolutionary history of salivary glands and their role in the adaptive radiation of mammals. Evolutionary patterns emerged when we made interspecific comparisons across mammalian orders. Among the bats, there is a clear relationship between SD secretion and general categories of diet.

Androgen regulation of the cellular Distribution of the true tissue kallikrein mK1 in the submandibular gland of the mouse.

The kallikrein gene family encodes for at least four different proteases in the mouse submandibular gland (SMG): mK1 (true tissue kallikrein), mK9, mK13, and mK22. These enzymes and many other biologically active proteins are synthesized by the granular convoluted tubule (GCT), a specialized segment of the SMG duct system. The GCT is under multihormonal regulation by androgens, thyroid hormones, and adrenocortical hormones. Androgens suppress synthesis of mK1 in the SMG but enhance expression of the other three kallikreins. We prepared an antibody with limited immunoreactivity for mK1 and used it to examine the effects of androgen status on the distribution of this isozyme in the SMGs of developing and mature mice by immunoperoxidase staining for the light microscope and immunogold labeling for the electron microscope. In prepubertal mice, every immature GCT cell contains mK1, confined to an accumulation of small granules in the subluminal cytoplasm. In mature mice, not every GCT cell contains mK1, and in those cells that do there is considerable intergranular variation in the intensity of staining for mK1. GCT cells containing mK1 are much more abundant in the glands of females than of males, resulting in a peculiar sexually dimorphic mosaic distribution of this isozyme in the mature SMG. Castration of adult males increases the number of GCT cells expressing mK1. Administration of androgen to intact or castrated males or to intact females reduces the number of cells staining for mK1. In all cases, immunogold labeling for mK1 is confined to secretory granules. No fine structural differences were noted between cells that were positively or negatively stained for mK1. Therefore, although GCT cells appear to be composed of a uniform population of cells on the basis of morphology alone, they are not homogeneous in their content of secretory proteins. These results indicate that androgen regulation of GCT cells is more complex than has been appreciated to date.

Ultrastructural study of hormonally responsive striated duct cells in the mouse sublingual gland.

In semithin sections stained with Heidenhain's iron hematoxylin, a few scattered granular cells were observed in the striated ducts (SDs) of sublingual glands (SLGs) of the mouse; they were seen normally only in the glands of adult males. However, it was shown by electron microscopy that many SD cells, other than these granular cells, had apical secretory granules, thus forming a granular striated tubule (named the GST in this study) in a portion of SD segments in both sexes. Sublingual GST cells had very small dense secretory granules near the apical surface, with the nucleus in the apical one-third to one-half of the cell; small Golgi apparatus; sparse rough endoplasmic reticulum (RER); and well-developed basal infoldings. However, some granular cells in male GSTs had abundant large dense secretory granules in the apical two-thirds of the cell, a basal nucleus, and modest basal infoldings. Such granular SD cells disappeared after castration in males. Granular SD cells could be induced in the GSTs of females by the injection of 5 alpha-dihydrotestosterone (DHT), triiodothyronine (T(3)), and/or dexamethasone (Dex); given simultaneously, these hormones acted synergistically in this induction. These results indicate a close similarity between the duct systems of the SLG and those of the submandibulan gland (SMG) of the mouse: granular SD cells of the GST in the SLG resemble GCT cells in the SMG in expressing some of the same biologically active polypeptides, in being sexually dimorphic, and in being under the same multihormonal regulation.

Transduction of TAT-HA-beta-galactosidase fusion protein into salivary gland-derived cells and organ cultures of the developing gland, and into rat submandibular gland in vivo.

We have studied the transduction of TAT-HA-beta-galactosidase fusion protein into two cell lines of rat salivary gland origin, A5 and C6-21, into cells of fetal mouse submandibular glands in organ culture, and into rat submandibular gland after retrograde duct injection, using a histochemical method to demonstrate beta-galactosidase activity. Transduction of the fusion protein into A5 and C6-21 cells was concentration- and time-dependent. Therefore, the intensity of the beta-galactosidase staining, which was cytoplasmic, was less after 1 hr of exposure compared to exposures up to 24 hr. However, the fusion protein was transduced into 100% of both types of cultured cells. When explants of mouse fetuses at 13 days of gestation were exposed to the fusion proteins, both epithelial and mesenchymal cells were stained for the enzyme, with a conspicuous accumulation of the reaction product at perinuclear cytoplasmic regions. The histochemical staining of the mesenchymal cells was more intense compared to that seen in epithelial cells. TAT-HA-beta-galactosidase fusion protein was also delivered to rat submandibular glands by retrograde duct injection. Histochemical staining for beta-galactosidase activity of cryostat sections prepared from the injected glands revealed that the transduction of the fusion protein was also time- and dose-dependent. In the glands of rats sacrificed from 10 min to 1 hr after the retrograde injection, essentially all acinar and duct cells showed cytoplasmic staining. The intensity of the staining then declined, and was not seen in the glands of rats killed 24 hr after the injection of the fusion proteins. These results indicate that a full-length, active TAT fusion protein can be targeted to salivary gland cells both in vitro and in vivo to analyze physiological, developmental, and pathophysiological processes.

Intracellular signalling cascades activated by the EGF receptor and/or by integrins, with potential relevance for branching morphogenesis of the fetal mouse submandibular gland.

The fetal mouse submandibular gland grown in vitro is a valuable model system to study epithelio-mesenchymal interactions vital for branching morphogenesis. This basic developmental process is dependent upon simultaneous engagement of receptors for growth factors and components of the extracellular matrix. Here we review intracellular signalling cascades mediated by activation of the EGFR in the developing mouse submandibular gland.

The ERK-1/2 signaling pathway is involved in the stimulation of branching morphogenesis of fetal mouse submandibular glands by EGF.

We have previously reported that epidermal growth factor (EGF) stimulates branching morphogenesis of the fetal mouse submandibular gland (SMG) (M. Kashimata and E. W. Gresik, 1997, Dev. Dyn. 208, 149-161) and that the EGF receptor (EGFR) is localized principally, if not exclusively, on the epithelial components of the fetal SMG (E. W. Gresik, M. Kashimata, Y. Kadoya, R. Mathews, N. Minami, and S. Yamashina, 1997, J. Histochem. Cytochem. 45, 1651-1657). The EGFR is a receptor tyrosine kinase, and after binding of its ligand, it triggers several intracellular signaling cascades, among them the one activating the mitogen-activated protein kinases (MAPK) ERK-1/2. Here we investigated whether EGF utilizes the ERK-1/2 signaling cascade to stimulate branching morphogenesis in the fetal mouse SMG. SMG rudiments were collected as matched pairs at E14, E16, and E18 (E0 = day of vaginal plug); placed into wells of defined medium (BGJb); and exposed to EGF for 5 or 30 min or to medium alone (controls). By Western blotting we found that EGF induced the appearance of multiple bands of phosphotyrosine-containing proteins, including bands at 170 kDa and 44 kDa/42 kDa, presumably corresponding to the phosphorylated forms of EGFR and ERK-1/2, respectively. Other blots showed the specific appearance of the phosphorylated EGFR and of phospho-ERK-1/2 in response to EGF. Immunohistochemical staining for phosphotyrosine increased at the plasma membrane after EGF stimulation for 5 or 30 min. Diffuse cytoplasmic staining for MEK-1/2 (the MAPK kinase that activates ERK-1/2) increased near the cell membrane after EGF stimulation. Phospho-ERK-1/2 was localized in the nuclei of a few epithelial cells after EGF for 5 min, but in the nuclei of many cells after EGF for 30 min. PD98059, an inhibitor of phosphorylation and activation of MEK-1/2, by itself inhibited branching morphogenesis and, furthermore, decreased the stimulatory effect of EGF on branching. Western blots confirmed that this inhibitor blocked phosphorylation of ERK-1/2 in fetal SMGs exposed to EGF. These results show that components of the ERK-1/2 signaling cascade are present in epithelial cells of the fetal SMG, that they are activated by EGF, and that inhibition of this cascade perturbs branching morphogenesis. However, EGF did not cause phosphorylation of two other MAPKs, SAPK/JNK or p38MAPK, in fetal SMGs. These results imply that the ERK-1/2 signaling is responsible, at least in part, for the stimulatory effect of EGF on branching morphogenesis of the fetal mouse SMG.

An unusual sexually dimorphic mosaic distribution of a subset of kallikreins in the granular convoluted tubule of the mouse submandibular gland detected by an antibody with restricted immunoreactivity.

The granular convoluted tubule of the mouse submandibular gland contains a wide variety of biologically active proteins, including several kallikreins. The tubule is under multihormonal regulation, and is sexually dimorphic, being larger in males than in females. Correspondingly, levels of its various protein secretory products are more abundant in males than in females. However, isoelectric focussing studies show that the true tissue kallikrein, mK1, is more abundant in the female than in the male submandibular gland. In this study, an antiserum was prepared with restricted immunoreactivity for mouse mK1, and possibly other kallikrein family members of low abundance in the mouse submandibular gland, and used for the immunocytochemical staining of the granular convoluted tubule cells in the submandibular gland of adult male and female mice, by indirect enzyme-labeled and immunogold-labeled antibody methods for light and electron microscopy, respectively. The distribution of immunoreactive tubule cells showed an unusual sexual dimorphism. In males only a few scattered slender tubule cells were strongly stained, while the more typical large tubule cells were only occasionally weakly positive, and many of them were not stained. By contrast, in females slender tubule cells were not seen, and about two thirds of the more typical tubule cells showed moderate to strong immunostaining. Immunoelectron microscopy revealed that immunostaining was confined to the secretion granules in granular convoluted tubule cells in both sexes. The slender tubule cells of males had many strongly stained small apical secretion granules and occasional basal infoldings; in the weakly positive larger more typical tubule cells not all secretion granules were positive, and there was intergranular variation in the intensity of staining of positive granules. In females, although more tubule cells were stained, intergranular variations in staining intensity were also noted. In both sexes, many tubule cells did not contain any secretion granules that showed immunogold labeling for kallikreins. These findings establish that, in contrast to the situation for the majority of granular convoluted tubules proteins, mK1 and possibly other minor kallikrein family members are more abundant in the granular convoluted tubules of female mice, and that there is considerable variation in the content of these kallikreins not only between different tubule cells, but also in individual secretion granules in any given tubule cell in either sex.

The EGF system in fetal development.

Immunohistochemical localization of the epidermal growth factor receptor (EGFR) disclosed a wide distribution of this protein in diverse fetal tissues. This review focuses on some of the vast work on the expression and operation of the EGFR and its principal ligands in fetal organogenesis.

Epidermal growth factor system is a physiological regulator of development of the mouse fetal submandibular gland and regulates expression of the alpha6-integrin subunit.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) regulate branching morphogenesis of fetal mouse submandibular gland (SMG) rudiments in vitro. The EGF system (EGF, TGF-alpha, and their shared receptor, EGFR) also regulates expression of integrins and their ligands in the extracellular matrix. We show here that inhibition of EGFR tyrosine-kinase activity by a tyrphostin retards in vitro development of SMGs. Using total RNA isolated from pooled SMGs taken from intact mouse fetuses, mRNA transcripts for EGF, TGF-alpha, and EGFR were detected by reverse transcription-polymerase chain reaction (RT-PCR), and age-dependent variations in the levels of these mRNA were quantitatively determined by nuclease protection assays. These findings suggest that the EGF system is operative in the in vivo development of this gland. alpha6-Integrin subunit was localized by immunofluorescence at the basal surface of epithelial cells. Branching morphogenesis of cultured SMG rudiments was inhibited by anti-alpha6 antibodies. Synthesis of alpha6-subunit in cultured SMGs, detected by metabolic labeling and immunoprecipitation, was increased by EGF and drastically reduced by tyrphostin. RT-PCR revealed that mRNAs for alpha6- and beta1- and beta4-integrin subunits are expressed at all ages between embryonic day 13 and postnatal day 7. These findings suggest that 1) the EGF system is a physiologic regulator of development of fetal mouse SMG, and 2) one mechanism by which it acts may be by regulating expression of integrins, which in turn control interaction of epithelial cells with the extracellular matrix.

Expression of epidermal growth factor receptor in fetal mouse submandibular gland detected by a biotinyltyramide-based catalyzed signal amplification method.

Branching morphogenesis of the fetal mouse submandibular gland (SMG) can be modulated in vitro by stimulation or inhibition of the epidermal growth factor receptor (EGFR). Because the mRNAs for EGF and EGFR are detectable in RNA of SMG rudiments isolated directly from fetuses, the EGF system probably operates physiologically as a regulator of SMG morphogenesis. However, neither EGFR protein nor its precise cellular localization has been characterized in the fetal SMG. Here we show EGFR protein in fetal mouse SMG by immunoprecipitation, affinity labeling, ligand-induced autophosphorylation, and immunohistochemistry. SMGs from E16 fetuses (day of vaginal plug = E0) were labeled with [35S]-cysteine/methionine and homogenized. After addition of specific antibody to EGFR, the immunoprecipitate was isolated, resolved by polyacrylamide gel electrophoresis, and detected by autoradiography. A single band of 170 kD was detected, corresponding to the EGFR protein. Affinity labeling with [125I]-EGF of the membrane fraction of E18 SMG also revealed a prominent band at 170 kD, showing that this EGFR protein can bind specifically to its ligand. Incubation of SMG membranes from E18 fetuses with EGF in the presence of [gamma-32P]-ATP, followed by immunoprecipitation with anti-phosphotyrosine antibody also showed a single band at 170 kD, demonstrating autophosphorylation of the EGFR in response to binding of its ligand. Immunohistochemical localization of the cellular sites of EGFR in the fetal SMG required use of a catalyzed signal amplification procedure, with biotinyltyramide as the amplifying agent. EGFR was localized predominantly, if not exclusively, in cell membranes of epithelial cells of the rudiment, whereas staining of mesenchymal cells was equivocal. Staining was strongest on duct cells, and weak on cells of the end-pieces. These findings clearly show that a functional EGFR protein is expressed in fetal SMG chiefly, if not exclusively, on epithelial cells.

Contemporary approaches to the study of salivary gland morphogenesis.

The submandibular gland of the mouse and rat serves as an important model to study epitheliomesenchymal interactions, since it can be studied both in vivo and in vitro. Previous work has established that the extracellular matrix, and especially the basal lamina, play vital roles in the development of this gland. More recent studies have indicated that integrins and several oncogene-related products, including components of the epidermal growth factor system, also play important roles in this gland's development.

The rodent granular convoluted tubule cell--an update.

The cells of the granular convoluted tubule (GCT) of the rodent submandibular gland (SMG) are under complex developmental and multihormonal regulation. Recent findings indicate that GCT cells also synthesize transforming growth factor alpha (TGF-alpha), hepatocyte growth factor, erythroid differentiation factor, endothelin, and insulin-like growth factor, as well as several novel androgen-dependent proteins of unknown function. The GCTs of hypophysectomized mice provide a convenient model to study multihormonal regulation of gene expression. The GCT system of the rodent SMG also is a fruitful model for study of hormone receptors.

Regulation of Na+,K+-ATPase in submandibular glands of hypophysectomized male mice by steroid and thyroid hormones.

The effects of thyroid hormone, androgen, glucocorticoid, and mineralocorticoid on Na+,K+-ATPase activity and on levels of its alpha-subunit protein (alpha 1 isoform) in mouse submandibular gland (SMG) were studied by enzyme assay for ouabain-sensitive ATP hydrolysis, by quantitative densitometric scanning of Western blots, and by immunohistochemistry. To define the specific regulatory effects of various pituitary-dependent hormones on expression of Na+,K+-ATPase in the SMG, we treated hypophysectomized (hypox) male mice with triiodo-L-thyronine (T3), 5 alpha-dihydrotestosterone (DHT), dexamethasone (Dex), and aldosterone (Ald), injected singly or in combination. Na+,K+-ATPase was confined to the duct system of the SMG. In intact mice there was a gender difference in SMG Na+,K+-ATPase, with levels of the enzyme's activity and of its alpha 1-subunit being less in the glands of males. In males, hypophysectomy caused a rise in levels of Na+,K+-ATPase activity and in levels of the alpha 1-subunit protein of this enzyme, and in intensity of immunocytochemical staining for this subunit but there were no such changes in the SMG of hypox females. Changes caused by hormonal replacement to hypox males in Na+,K-ATPase activity, levels of its alpha 1-subunit, or the intensity of immunocytochemical staining for this subunit were complex. Ald had no effect. T3 or dexamethasone, given alone, induced Na+,K+-ATPase activity above control values (hypox males) and increased levels of its alpha 1-subunit protein and immunohistochemical staining for this subunit. By contrast, DHT did not cause a decline in any of these parameters. However, when treatment with T3 was combined with administration of Dex or DHT, enzymatic activity of Na+,K+-ATPase decreased but levels of the alpha 1-subunit protein and immunohistochemical staining for this subunit increased. Therefore, inductions of the alpha 1-subunit of this enzyme are not always correlated with increases in levels of activity of Na+,K+-ATPase, and we propose that both enzymatic and immunochemical analyses are essential for evaluation of hormonal regulation of Na+,K+-ATPase in salivary gland and in other tissues.

The granular convoluted tubule (GCT) cell of rodent submandibular glands.

The granular convoluted tubule (GCT) is a segment of the duct system of all rodents, situated between the striated and intercalated ducts. It has the peculiar property of synthesizing a large variety of biologically active polypeptides whose role in saliva remains unknown. The literature on the fine structure of GCT cells is critically reviewed. Some recent developments on endocrine regulation of the structure and contents of rodent GCT cells are summarized, with emphasis on EGF, NGF, renin, and kallikrein proteases. A survey of the distribution of GCT cells in several vertebrate families is presented.

Effects of female hormones and 3,5,3'-triiodothyronine or dexamethasone on induction of epidermal growth factor and proteinases F, D, A, and P in the submandibular glands of hypophysectomized male mice.

The effects of progesterone (Pro), 5 alpha-dihydrotestosterone (DHT), 17 beta-estradiol (E2), T3, and dexamethasone (Dex), given alone or in combination, on induction of epidermal growth factor (EGF) and proteinase isozymes in the submandibular glands of hypophysectomized mice were examined. Each hormone, except E2, acting alone had essentially comparable inductive effects on EGF concentrations and on total proteinase activity. E2 alone had no inductive effect at all. Pro acted synergistically with T3, but its inductive effect was diminished when given with Dex. E2 was not synergistic with T2, and it inhibited the effect of Dex; it also partially blocked the action of DHT on induction of proteinase activity but not of EGF. Simultaneous administration of all five hormones restored total proteinase activity completely but EGF levels to only 50% of values for intact males, respectively. Submandibular proteinases were resolved by isoelectric focusing into four isozymes: proteinase F (pI 4.8), proteinase D (pI 5.8), proteinase A (pI 6.2), and proteinase P (pI 10.0). Pro alone slightly increased levels of proteinase F but greatly raised levels of the other three isozymes. This inductive action was augmented when Pro was given with T3 but blocked when it was given with Dex. E2 alone not only failed to induce any of the isozymes, but even further reduced levels of proteinase F. It also decreased the inductive effects of T3 on these isozymes, and with Dex completely blocked induction of proteinases D, A, and P. E2 plus DHT suppressed proteinase F levels and only induced proteinase A. All five hormones together reestablished the isozyme profile seen in intact males. These results show that Pro by itself is as capable as androgens, thyroid hormone, or glucocorticoid in regulating expression of these submandibular polypeptides, and that its action can be modulated by other pituitary-dependent hormones. In addition, they demonstrate that E2 does not regulate their expression, and that it has an inhibitory effect on the inductive action of other hormones. Last, they indicate that these various hormones may regulate expression of EGF and of each of the individual proteinase isozymes differently.

Additive and/or synergistic effects of 5 alpha-dihydrotestosterone, dexamethasone, and triiodo-L-thyronine on induction of proteinases and epidermal growth factor in the submandibular gland of hypophysectomized mice.

The effects of androgen, glucocorticoid, and thyroid hormones on levels of proteinase isozymes and epidermal growth factor (EGF) in the submandibular glands of hypophysectomized (Hypox) mice were investigated. Total proteinase activity in males was decreased by hypophysectomy and increased by single or combined injection of the three hormones into these mice. 5 alpha-Dihydrotestosterone (DHT) had the strongest effect, and dexamethasone (Dex) the least. By isoelectric focusing, proteinases extracted from the submandibular gland of untreated male and female mice were fractionated into four isozymes with pI values of 4.8 (proteinase-F), 5.8 (proteinase-D), 6.2 (proteinase-A), and 10.0 (proteinase-P). In Hypox mice (both sexes), there was only a single isozyme, proteinase-F. Proteinase-D, -A, and -P were induced in the submandibular gland of Hypox males by injections of DHT, Dex, and/or T3; the percent ratio of activity of each of these isozymes induced by these hormones, given either singly or in combination, was almost parallel among the three isozymes. Synergistic effects were observed between T3 and Dex, and additive effects between T3 and DHT. The increase in proteinase isozyme activities by concomitant injections of T3 and Dex was about 2 times more than the additive values. The changes in proteinase-F upon hormone injection were complicated. In females, the enzyme activity was decreased by hypophysectomy and increased by DHT administration. In males, on the other hand, it was increased by hypophysectomy and suppressed by T3 or T3 plus steroid hormones. The EGF level in the submandibular gland was decreased to about 1/800th (males) or 1/90th (females) of its normal level by hypophysectomy. Its level in the Hypox animal was greatly increased by all three hormones, given singly or in combination. Synergism was also observed between T3 and steroid hormones; DHT plus T3 and T3 plus Dex induced EGF 6 times and 9 times, respectively, more than the additive values. These values were much greater than those for the induction of proteinase-D, -A, and -P by combined injections of T3 and steroids. The present results suggest that the genes coding for proteinase-A, -D, and -P are located close to each other and that the onset of their expression may be controlled by the same regulatory mechanism. By contrast, the gene for proteinase-F may be mapped to a different locus or regulated differently. The mechanism of induction of EGF by T3, DHT, and Dex appears to be similar to but not completely the same as that for proteinase-D, -A, and -P.

Adrenergic regulation of c-fos expression in cultured BC3H1 muscle cells.

Administration of adrenergic agonists induced c-fos mRNA in the salivary glands of the mouse and in the heart of the mouse, rat, and hamster (Barka et al., 1986, Mol. Cell Biol. 6, 2984-2989; 1987; Oncogene 1, 439-443). To further analyze transcriptional and post-transcriptional control of c-fos expression by adrenergic receptors and the putative role of fos in replication and differentiation pathways, we have examined c-fos expression in BC3H1 cells, a tumor-derived nonfusing muscle cell line. BC3H1 cells possess alpha 1- and beta 2-adrenergic receptors as well as receptors for histamine and acetylcholine. Furthermore, rapidly proliferating BC3H1 cells undergo differentiation toward muscle phenotype when exposed to low serum-containing culture media. Both alpha- and beta-adrenergic agonists and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate caused a rapid, transient increase in the steady-state level of c-fos mRNA. This induction was essentially independent of whether the cells were in the proliferative, relatively quiescent, or differentiated state. Protein synthesis inhibitors cycloheximide and anisomycin also increased markedly the concentration of c-fos mRNA, and in the presence of anisomycin c-fos mRNA was superinduced by the alpha-adrenergic agonist norepinephrine. Run-on transcription assays indicated that the c-fos gene is expressed in both proliferating and differentiated cells, although the steady-state levels of c-fos mRNA were low, or even undetectable, in such cells. The adrenergic agonists and the tumor promoter stimulated the transcription of the c-fos gene in both proliferating and differentiated cells. This stimulation, however, was modest, two- to three-fold compared to controls, in contrast to the marked elevation of the level of c-fos mRNA they caused. Neither the proliferation nor the expression of muscle type creatine kinase activity was influenced by adrenergic agonists. It is suggested that activation of the c-fos gene is a consequence of adrenoreceptor stimulation in diverse cell types, and thus it is involved in pleiotropic cellular responses to adrenergic agonists. Catecholamines may be one of the physiologic regulators of the c-fos gene.

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.

Autoradiographic localization of dihydrotestosterone binding in the major salivary glands and other androgen-responsive organs of the mouse.

Mouse submandibular glands show an androgen-dependent sexual dimorphism, reflected in higher concentrations in males than in females of bioactive peptides, such as epidermal growth factor (EGF), nerve growth factor, and renin in the cells of the granular convoluted tubules (GCT). Biochemical studies have demonstrated androgen receptors in submandibular gland and other androgen-responsive organs in mouse. We have determined the cellular localization of these receptors using steroid autoradiography. Fifteen adult gonadectomized male mice were injected intravenously with 0.13 microgram or 0.26 microgram [3H]-dihydrotestosterone (SA 135 Ci/mM); some animals were pre-treated with cyclocytidine to stimulate secretion by GCT cells. Animals were killed 15 min, 1, 2, or 3 hr after isotope injection. Steroid autoradiographs were prepared, and some were stained immunocytochemically for EGF. Of the different cell types of submandibular gland, the acinar cells most frequently and intensely concentrated [3H]-DHT; GCT cells also concentrated the hormone, as did a small number of striated duct cells. In the other major salivary glands, the only cells that concentrated the androgen were interlobular striated duct cells in sublingual gland. In prostate, anterior pituitary, and brain a large number of cells concentrated androgen, as has been previously reported. Androgen binding by the GCT cells was a predictable finding, since androgen-induced alterations in composition and form of these cells are well documented. The intense androgen concentration by the acinar cells was an unexpected finding and suggests a hitherto unknown androgen regulation of these cells. An incidental finding was intense concentration of [3H]-DHT in the nuclei of the endothelial cells of the post-capillary venules of the cervical lymph nodes.

Inductive effects of triiodothyronine or dihydrotestosterone on EGF in the submandibular glands of young, middle-aged, and old C57BL/6NNia female mice.

Effects of advancing age on responsiveness to triiodothyronine (T3) or dihydrotestosterone (DHT) by granular convoluted tubule (GCT) cells in submandibular glands were studied in C57BL/6NNia female mice of 4, 12, and 24 months of age. In untreated mice, absolute GCT volume and the frequency of cells strongly immunostained for EGF increased with age. Concentrations of EGF rose fourfold. Both T3 and DHT had inductive effects at each age: GCT cells were enlarged, contained more granules, and stained very strongly for EGF; EGF concentrations and the volume of GCTs also increased. However, the pattern of induction differed with age for the two hormones. After T3 treatment EGF concentration declined with age with respect to the degree of induction over same age control levels. By contrast, after DHT treatment the level of induction of EGF over control levels was the same at all three ages. The pattern of aging of GCT cells and their responsiveness to T3 or DHT in female mice differ from that previously reported for males.