Beta-adrenergic stimulation of c-fos gene expression in the mouse submandibular gland.

Isoproterenol (IPR), a beta-adrenergic agonist, induces division of acinar cells in the parotid and submandibular glands of adult rodents and produces hyperplastic and hypertrophic enlargements of these organs. We analyzed the effects of IPR on thymidine incorporation, c-fos mRNA levels, and the immunocytochemical localization of c-fos protein in the submandibular glands of adult and of 5- and 14-day-old mice. In the glands of untreated mice c-fos transcripts were not detectable. In all experimental groups, administration of IPR led to a rapid, transient increase in the c-fos mRNA level. Propranolol blocked the IPR effect, while treatment with IPR and cycloheximide led to superinduction. We observed no correlation between the effect of IPR on cell replication or organ growth and stimulation of c-fos expression, and conclude that the latter is the result of beta-adrenergic receptor-IPR interaction. The c-fos protein was localized immunocytochemically in both the cytoplasm and the nuclei of acinar cells and in the nuclei of duct cells.

Effects of isoproterenol on mammary gland tumors induced by N-nitroso-N-methylurea and salivary gland tumors induced by 7,12-dimethylbenz[a]anthracene.

The effect of isoproterenol (IPR) (20 mg/kg, twice per wk) on mammary gland tumors induced by N-nitroso-N-methylurea (NMU) (40 or 77 mg/kg, iv) was studied. Within 18 weeks 50--60% of the noninbred female Sprague-Dawley rats that received a single injection of NMU developed carcinomas of the mammary gland. In addition, a malignant lymphoma was observed. There was no change in the incidence of tumors if NMU was administered at the time that DNA synthesis was stimulated in the submandibular glands by two injections of IPR (160 mg/kg, ip) given 24 hours apart. Tumors of unequivocal salivary gland origin were not observed, irrespective of whether NMU was given at the peak of stimulated DNA synthesis or without pretreatment with IPR. However, in 10% of the tumor-bearing rats, tumors were found in the neck region. These tumors could be separated from the salivary glands by dissection and were of mammary gland origin. Chronic treatment with IPR caused marked enlargements of the parotid and submandibular glands with hypertrophy of the acinar cells and degeneration of the duct system. Such a treatment caused a high death rate but no change in the incidence of the tumors. The effect of chronic IPR treatment (10 mg/kg twice per wk or 5 mg/kg three times per wk) on submandibular gland tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) was also studied. With the intraglandular injection of 100 or 200 micrograms of DMBA, the incidences of squamous cell carcinomas in the glands were 6.8 and 38.6%, respectively. Chronic administration of IPR after the instillation of DMBA caused a high death rate but no statistically significant change in the incidence of salivary gland tumors.

Proto-oncogene fos (c-fos) expression in the heart.

Administration of the beta-adrenergic agonist isoproterenol led to a marked rapid increase in the steady-state level of c-fos mRNA in the heart of mice, rats, and Syrian hamsters. Stimulation of c-fos expression by isoproterenol was inhibited by the beta-adrenergic antagonist propranolol. An increase in Ca2+ influx through voltage-dependent calcium channels is probably not required for the activation of the c-fos gene by isoproterenol since the calcium channel blockers verapamil, nifedipine, and diltiazem had no effect on the induction of c-fos by the drug. In the heart of the rat, c-fos expression was also stimulated by the alpha-adrenergic agonist phenylephrine, histamine, and prostaglandin E1. The histamine-induced expression of the c-fos gene was blocked by the histamine H1-receptor antagonist pyrilamine but not by H2-receptor antagonists ranitidine and cimetidine. It is concluded that in the heart, hormones which increase cAMP and cytosolic Ca2+, such as beta-adrenergic agonists and prostaglandin E1, and/or stimulate the turnover of inositol phospholipids, such as alpha-adrenergic agonists and histamine H1-receptor agonists, regulate c-fos gene expression. The fos protein is likely to play a role in the mechanisms of neurotransmitters and hormones that modulate the functioning of the heart and of cardiac hypertrophy, degeneration and necrosis.

Accumulation of c-fos mRNA in slices of mouse submandibular gland incubated in vitro.

Incubation of slices or isolated lobules of murine submandibular gland at 37 degrees C in physiologic solutions or in tissue culture media, or dissociation of the cells by collagenase-hyaluronidase treatment, increased the steady-state level of c-fos mRNA without any additional stimulus. This activation of c-fos expression required the presence of Na+ and K+ but not extracellular Ca2+. It was augmented by depolarizing concentrations of K+ and by veratridine, and inhibited by high concentrations of amiloride. Alterations in membrane permeability and in ion fluxes and/or perturbation in membrane phospholipids may play a role in this transitional activation of the c-fos gene expression in incubated tissue slices in which the cells are not viable and undergo a necrobiotic process.

Beta-adrenergic receptors and adenylate cyclase in hypertrophic and hyperplastic rat salivary glands.

Isoproterenol induces both the secretion of protein and the stimulation of DNA synthesis and growth in rat salivary glands. The specific binding of the labelled beta-adrenergic antagonist [3H]dihydroalprenolol has been used to measure the number of beta-adrenergic receptors in rat parotid glands during isoproterenol-induced growth. Isoproterenol-enlarged glands display no change in the specific binding capacity per gland for [3H]-dihydroalprenolol compared with normal tissue. Catecholamine sensitive adenylate cyclase activity varies independently of the number of specific [3H]dihydroalprenolol binding sites during isoproterenol-induced growth. Previously-described di-ferences in optimal isoproterenol doses which produce protein secretion and stimulation of DNA synthesis may reflect different responses to various rates of receptor occupancy, or may be due to the presence of more than one type of beta-adrenergic receptor.

Retrovirus-mediated gene transfer into salivary glands in vivo.

In the present report, we show prolonged expression of beta-galactosidase (beta-Gal) in the acinar cells of the submandibular and sublingual glands of rats following retrograde ductal injection of the retroviral vector BAG. To facilitate integration of viral DNA, cell division in the gland was induced by the administration of the beta-adrenergic agonist isoproterenol prior to the delivery of the vector. The frequency of cells stained for beta-Gal was higher if the virus was injected 4-20 hr after the two injections of isoproterenol given 24 hr apart than after the injection of only one dose of the drug. Without stimulation of cell division, no integration of the viral DNA was observed. Expression of the marker enzyme was observed up to 43 days, the limit of the observation period. The data indicate that salivary glands are potential targets of retrovirus-mediated gene transfer for somatic gene therapy.

Hormonal regulation of epidermal growth factor and protease in the submandibular gland of the adult mouse.

The structure of the granular convoluted tubules of the mouse submandibular gland is influenced by androgens, adrenal steroids, and thyroid hormones. We wished to investigate the effects of variations in hormonal status on the quantitative and qualitative distribution of two secretory products of these tubules, epidermal growth factor (EGF) and protease. The effects of the thyroid and adrenal glands on EGF content and protease activity of the submandibular glands of adult female mice were studied by RIAs (EGF), enzyme assays (protease), and immunocytochemical methods. In animals rendered chronically hypothyroid by propylthiouracil (4 months) or in animals which were adrenalectomized and ovariectomized (3 weeks), protease activity and EGF levels were reduced by 81-97%. The administration of testosterone induced these polypeptides even in hypothyroid animals. Daily administration of L-T4 (T4; 1 micrograms/g BW) for 7 days increased EGF and protease activity 3.6-fold in intact mice and reversed the effect of hypothyroidism. EGF and protease were also induced by T4 in adrenalectomized and ovariectomized mice, although to a lesser degree than in intact animals. Immunocytochemical stainings of submandibular glands indicated that the number of granular convoluted tubule cells immunoreactive for EGF correlated with the levels of EGF determined by RIAs. With respect to immunostaining for protease, such a correlation was not observed. The data indicate multihormonal regulation of EGF and protease in the mouse submandibular gland.

Epidermal growth factor gene expression is regulated differently in mouse kidney and submandibular gland.

The concentration of the peptide mitogen epidermal growth factor (EGF) is hormonally and developmentally regulated in the granular convoluted tubule cells of the mouse submandibular gland. Using a labeled EGF nucleic acid probe, we have demonstrated that submandibular gland EGF mRNA concentrations increase during postnatal development of the gland and after the administration of testosterone or thyroid hormone. Recently, it was reported that EGF mRNA is present in kidney as well as a number of other mouse tissues. A comparison of EGF gene regulation in submandibular gland and kidney revealed that kidney EGF mRNA levels also increase during the postnatal period. Opposite sex differences were observed, with submandibular gland levels being about 16-fold higher in the male than in the female and kidney levels being 2- to 4-fold higher in the female than in the male. Renal EGF mRNA concentrations are less responsive to hormones than those in the submandibular gland. Renal EGF was localized immunocytochemically to the cells of distal convoluted tubules.

Biologically active polypeptides in submandibular glands.

Since the discovery of kallikreins in the submandibular glands in 1963 by Werle and Roden, a great number of biologically active polypeptides has been purified from, or claimed to be present in, the submandibular of the mouse and of other species. In this review, available data on the occurrence, chemical properties, localization, hormonal control, synthesis, secretion, and possible physiologic roles of 25 biologically active factors in mouse submandibular gland are analyzed. In general, these factors are androgen dependent, but not affected by the sex genotype, and are localized in the granular convoluted tubule cells in the gland. They are secreted into the saliva, but are also found in the circulation. Their physiologic roles are largely unknown.

Distribution of concanavalin A binding sites on the surface of dissociated rat submandibular gland acinar cells.

The submandibular glands of 4-week-old rats were dissociated by a procedure involving digestions with collagenase and hyaluronidase, chelation of divalent cations and mechanical force. A suspension of single cells was obtained in low yield by centrifugation in a Ficoll-containing medium. Immediately after dissociation and after a culture period of 16-18 hr the dissociated cells were tested for agglutinability by concanavalin A (Con A). Using ferritin (tfer)-conjugated Con A the lectin binding by the isolated acinar cells was also studied. The dissociated cells were agglutinated by low concentrations of Con A and bound Fer-Con A molecules on their entire surface without any indication of polarization of the cell membrane. There was a considerable cell to cell variation in the amount of Fer-Con A binding which was, in general, sparse and patchy. The contact surfaces between agglutinated cells revealed a dense binding of Fer-Con A molecules irrespective of the types of cells participating in the agglutination reaction. Cells cultured for 16-18 hr were no longer agglutinated by Con A. As compared to the freshly dissociated cells the cultured acinar cells revealed a more uniform and denser binding of Fer-Con A molecules. Furthermore, there were more lectin molecules bound to the cell surface corresponding to the basal part of the cell, where the nucleus and most of the rough surface endoplasmic reticulum were located, than to the apical cell surface. It is suggested that the higher density of lectin-binding sites on the cell surface in the vicinity of the cisternae of the rough endoplasmic reticulum indicates insertion sites of newly synthesized membrane glycoproteins.

Immunocytochemical localization of epidermal growth factor in mouse submandibular gland.

The cellular and subcellular localization of epidermal growth factor in the submandibular glands of male and female adult mice was established by immunoperoxidase techniques. In light microscopic preparations epidermal growth factor was found exclusively in the granular convoluted tubules of the gland. The intensity of staining for epidermal growth factor varied from cell to cell, and some cells apparently were negative. The pattern of staining was similar in the glands of male and female mice; however, the granular convoluted tubules are androgen-responsive, and thus more extensive and composed of larger cells in males. In thin sections epidermal growth factor was most heavily concentrated in the secretion granules of the granular convoluted tubule cells. Within a given cell there was variation in intensity of staining of individual secretion granules, with some granules appearing minimally reactive or negative. The only other cell component with deposits of reaction product was the ribosomes.

Expression of the cysteine proteinase inhibitor cystatin C gene in rat heart: use of digoxigenin-labeled probes generated by polymerase chain reaction directly for in situ and northern blot hybridizations.

Cystatins represent a widely distributed superfamily of cysteine proteinase inhibitory proteins. We investigated the expression of the cystatin C gene, belonging to the family 2 of cystatins, in the hearts of female rats. Using a highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) we have detected cystatin C mRNA in the ventricule and atrium, as well as in liver and submandibular gland. A digoxigenin-labeled cystatin C probe, generated by PCR, hybridized to a single mRNA species of about 700 nucleotides on Northern blots. Northern blot hybridizations established that neither an acute inflammation produced by injection of turpentine nor administration of the beta-adrenergic agonist isoproterenol had an effect on the level of cystatin C mRNA in the heart. In situ hybridizations with digoxigenin-labeled probe localized the expression of the cystatin C gene to cardiac muscle fibers but not to other cardiac cellular elements. Cystatin C may be released by cardiac muscle fibers under physiological and pathological conditions and may modify inflammatory and necrobiotic processes.

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.

Retrovirus-mediated gene transfer into rat salivary gland cells in vitro and in vivo.

A retroviral vector DAP that encodes the human placental alkaline phosphatase (PLAP) and the neomycin-resistant gene was used to transduce the salivary gland-derived cell line A5 in vitro and acinar cells in rat submandibular gland in vivo. Expression of the transduced PLAP gene was established by histochemical staining for heat-resistant AP and by determination of enzyme activity. From the in vitro experiments, we concluded that the salivary gland-derived cell line A5 can be infected by the retroviral vector DAP. In the transduced cells the viral long terminal repeat (LTR) promoter was effective, and the cells expressed heat-stable PLAP which was localized mostly in the plasma membrane and could be released by treatment with bromelain or phosphatidyinositol-specific phospholipase C. A5-DAP cells secreted PLAP into the medium. Clones of A5-DAP cells expressed various levels of the enzyme. The level of enzyme activity in different clones was unrelated to growth rate. Retrograde ductal injection of the viral vector into the duct of the submandibular gland of rats resulted in integration and long-term expression of PLAP gene in acinar cells. Expression of PLAP was seen up to 25 days, the limit of the observation period. To facilitate integration of the viral DNA, cell division of acinar cells was induced by administration of the beta-adrenergic agonist isoproterenol before administration of the virus. PLAP was secreted into submandibular saliva. The data support the notion that salivary glands are suitable targets for gene transfer in vivo by a retroviral vector.

Immunocytochemical localization of renin in the submandibular gland of the mouse.

Renin was localized in the submandibular gland of the adult mouse at light and electron microscopic levels by the unlabeled antibody enzyme method of Sternberger. At the light microscopic level, renin was confined to the granular convoluted tubule (GCT) segment of the gland with considerable variation among GCT cells in intensity of staining. Some GCT cells failed to stain for renin. The pattern of staining was the same in the gland of male and female mice, but in the glands of females GCT segments were smaller and less numerous. At the electron microscopic level, staining for renin was also confined to the GCT cells, and was localized exclusively to the secretory granules. The intensity of staining of the secretory granules within a given GCT cell varied; some cells contained only minimally reactive or negative secretory granules. All other organelles within the GCT cell, except condensing vacuoles, failed to stain.

Immunocytochemical investigations on the submandibular glands of developing and adult mice using a specific antiserum on protease A.

The submandibular glands of developing and adult mice were studied immunocytochemically by the unlabeled antibody peroxidase-antiperoxidase and the colloidal gold-protein A methods, using an antiserum to a highly purified esteroprotease (protease A, EC 3.4.4) of mouse submandibular gland origin. A thin subluminal rim of immunoreactivity, seen in striated duct cells throughout development, persisted in adulthood. From 15 days of age onwards, striated duct cells with diffuse cytoplasmic staining also occurred; such cells increased in number with age. A clear sexual dimorphism of the submandibular gland was first discernable by 25 days of age, when the developing granular convoluted tubule (GCT) cells of males were slightly larger than those of females; this size difference became more pronounced at later ages, resulting in a distinct dimorphism by 50 days of age. In adults, the principal sites of immunoreactivity were the GCTs, whose component cells stained with different intensities. Electron microscopic immunocytochemical techniques revealed that deposits of oxidized diaminobenzidine or particles of celloidal gold were restricted to the secretion granules of GCT cells; all other organelles were unstained. Acinar and intercalcated duct cells were negative.

Epidermal growth factor and renin in mouse submandibular glands.

Epon sections of the submandibular gland of SWF/J male mouse were stained immunocytochemically for epidermal growth factor (EGF) and renin. Most cells of the granular convoluted tubules (GCT) contained both EGF and renin. However, examinations of adjacent semithin or thin sections stained for EGF and renin, respectively, revealed a small population of GCT cells that contained EGF but no renin. Within a cell all secretory granules contained both EGF and renin. The renin-negative/EGF-positive cells may represent a subpopulation of tubular cells that do not express, or carry, the renin gene.

Immunocytochemical localization of amylase in the parotid gland of developing and adult rats.

An antiserum against purified rat parotid amylase was used to localize the protein in parotid glands of developing and adult rats. The unlabeled antibody peroxidase-antiperoxidase method and the protein A-gold colloid technique were used at the light and electron microscope levels, respectively. Immunoreactive amylase was detected in a few scattered cells in the glands of 2-day-old rats. During the following days the number of cells stained immunocytochemically for amylase increased rapidly; at 15 days of age all acinar cells revealed amylase, but the intensity of immunostaining varied from cell to cell. Electron microscopically, amylase was localized in the secretory granules, and by using a more concentrated antiserum, in the rough endoplasmic reticulum and Golgi complex. At early stages of development the acinar cells contained fewer and smaller secretory granules than in adult animals; the gold particles indicative of amylase were randomly distributed over the secretory granules. In the glands of adult rats, amylase was distributed inhomogeneously within the secretory granules. In the majority of secretory granules gold colloid particles were located over the electron-dense portions of the granules. However, secretory granules in which an amylase-rich shell surrounded an amylase-poor or amylase-negative "core" were not infrequent.

In situ localization of mRNA for epidermal growth factor in the submandibular gland of the mouse.

Epidermal growth factor (EGF) is a polypeptide originally isolated from the mouse submandibular gland, where it is localized immunocytochemically in cells of the granular convoluted tubules (GCT). cDNAs encoding the precursor of mouse submandibular EGF have been cloned (Scott et al. Science 221:236, 1983; Gray et al. Nature 303:722, 1983). A fragment of one of these clones, pmegf10, containing the EGF coding region, was tritium-labeled by nick-translation and used as a probe for in situ hybridization to EGF mRNA. A specific hybridization signal for EGF mRNA was seen only in mature or developing GCT cells. The intensity of the signal was stronger in glands of intact males than in females or in castrated males. In glands of castrates treated with testosterone, or of intact females treated with triiodothyronine (T3), the signal was comparable to that in intact males. In glands of males treated with T3 the intensity of the signal was stronger than in untreated males. A weak to moderate signal was seen in developing GCT cells of 20-day-old males but not females. Hybridization for 3 days gave a stronger signal than that for 1 day. No signal was seen in either sex at 10 days of age, or in control preparations exposed to labeled DNA of pBR322. The presence of EGF mRNA exclusively in GCT cells provides strong evidence that these cells are the only site of synthesis of EGF in the submandibular gland. In situ hybridization with this cDNA probe will provide a sensitive method to determine possible cellular sites of EGF production outside of the submandibular gland.

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.