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SCA-9 cell line was developed from an induced tumor of mouse submandibular gland. We have studied some of the phenotypic characteristics of SCA cells cultured on different matrices. On plastic surface, the cells grow as a monolayer; on matrigel, they form branching structures and tubes, a phenomenon termed branching morphogenesis. EGF and HGF promoted cellular growth and branching morphogenesis which was inhibited by anti-EGF antibodies. We have performed RT-PCR and real-time quantitative RT-PCR of cells grown on plastic surface or on matrigel. Grown on plastic, the cells express EGF and renin 2, but no or only trace amounts of NGF. Growth on matrigel for 24 h resulted in a transient 21-fold increase in EGF mRNA and a 3371-fold increase in renin 2 mRNA. There was no change in NGF mRNA level. SCA-9 cells express mRNAs for receptors for the EGF family of ligands. On plastic, mainly ErbB1 and ErbB2 are expressed. Culture on matrigel resulted in 11-fold increase in mRNA levels for ErbB1 and ErbB2, and a 221-fold and 85-fold increase in the mRNA levels for ErbB3 and ErbB4, respectively. Small interfering RNAs siErbB3 and siErbB4 inhibited the growth of the cells grown on plastic or matrigel. Significant growth inhibition was seen also with siErbB1+siErbB3 and siErbB2+siErbB3. siErbB1 and siErbB2 also inhibited branching morphogenesis. Since SCA cells express EGF and receptors for EGF, EGF acts an autocrine regulator in promoting growth and branching morphogenesis. We conclude that SCA cells provide a useful model to analyze the mechanism of branching morphogenesis and the role of matrix in regulating expression of phenotypic characteristics of cultured cells.

We have studied the transduction of TAT-HA-β-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 β-galactosidase activity. Transduction of the fusion protein into A5 and C6–21 cells was concentration- and time-dependent. Therefore, the intensity of the β-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-β-galactosidase fusion protein was also delivered to rat submandibular glands by retrograde duct injection. Histochemical staining for β-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.

We have studied the effects of sodium butyrate (NaBu) on the expression of genes transduced by retroviral vectors and stably expressed in two salivary gland-derived cell lines, A5-DAP and A5-BAG, established earlier. These cell lines were obtained by infecting A5 cells with the retroviral vectors DAP and BAG, respectively, and by selecting neomycin-resistant transduced cells. A5-DAP cells express human placental alkaline phosphatase (PLAP) and A5-BAG cells bacterial beta-galactosidase, both under the control of the viral long terminal repeat (LTR) enhancer-promoter. NaBu in the concentration of 2-8 mM inhibited the growth of A5-DAP cells, and induced the expression of heat-stable PLAP. These effects of NaBu were dose-dependent. Induction of PLAP in clones of A5-DAP cells that express different basal levels of the enzyme was not correlated with the relative inducibilty by NaBu. Exposure to 4 mM NaBu for 48 h increased the PLAP mRNA level by 31%. A5-DAP cells released, in a time-dependent manner, PLAP into the culture medium. Cells treated with NaBu released more PLAP than untreated cells in proportion to their elevated level of the enzyme. The parent A5 cells also express a low level of tissue non-specific type alkaline phosphatase, which was also induced by NaBu. NaBu inhibited the growth of A5-BAG cells also, and increased the beta-galactosidase level. These data indicate the genes transduced by retroviral vectors can be induced by NaBu, which most likely interacts with the viral LTR.

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.

Rat cystatin S and rat cystatin C are members of family 2 (cystatin) of the cystatin superfamily. All members of the cystatin family inhibit cysteine proteinases to varying degree. The expression of these two inhibitors, which have a 48% similarity at the nucleotide level, was studied in the submandibular gland using reverse transcriptase-polymerase chain reaction (RT-PCR). Northern blot hybridization and in situ hybridization with digoxigenin-labelled DNA probes. Both inhibitors were expressed in the serous acinar cells of the submandibular gland. In accord with previous findings, cystatin S mRNA was induced by the beta-adrenergic agonist isoproterenol. The level of cystatin S mRNA, which was very low in the glands of untreated rats and was demonstrable by RT-PCR but not by Northern blot hybridization, was not altered by acute inflammation produced by turpentine. Neither the administration of isoproterenol nor acute inflammation had any effect on the level of cystatin C mRNA, indicating beta-adrenoreceptors are not involved in the regulation of the cystatin C gene(s) in the submandibular gland. The data indicate that these two closely related genes, expressed in the same cells, are differently regulated. The consequence of this difference in gene regulation on the physiological and pathological roles of these inhibitors remains to be established.

PC12 rat pheochromocytoma cells were found to contain and release into the culture medium cysteine proteinase inhibitors, cystatins. Inhibitory activities in cell extracts and in spent culture media were assayed by titration of papain using a sensitive fluorescent method. Partial purification of the inhibitors using gel-exclusion and ion exchange chromatography and immunoblotting indicated that PC 12 cells contain high and low molecular weight cystatins. The low molecular weight (14 to 17 kDa) cystatins reacted with antibody against human cystatin C. Using the same antibody, cystatin-like immunoreactive material was localized in the cytoplasm of PC12 cells. Treatment of PC12 cells with sodium butyrate (6 mM), which is known to induce chromaffin cell-type differentiation, inhibited cell proliferation and led to a significant increase in cystatin level. The data suggest that cystatins in PC 12 cells may be involved in differentiation processes. The well-characterized PC12 cells, capable of neuronal or chromaffin cell differentiation, will provide a useful cell type to study the regulation of cystatin gene expression and the physiologic role (s) of cystatins.

The activities of cysteine proteinases which include several lysosomal cathepsins are controlled by naturally occurring inhibitory proteins termed cystatins. Cystatins occur both intracellularly and extracellularly in various tissue fluids including tears. Tears were collected by the Schirmer paper strip method from healthy volunteers who had no history or signs of external ocular disease. The tear components were extracted from the filter papers, and used to determine the apparent free cystatin activity and cystatin levels of tears, and for immunoblots. Tears were also collected using capillary tubes for the measurements of cystatins. By titrating papain, a cysteine proteinase, of known specific activity with tear fluid, relatively high levels of apparent free cystatin activity were demonstrated in tears: 28.8 +/- 3.47 (S.E.M.) pmols papain inhibited per mg tear protein (n = 9). The concentrations of cystatins in tear samples were measured by an indirect enzyme-linked immunosorbent assay (ELISA) using antibodies against human salivary cystatin S and purified cystatin S as standard. The ELISAS revealed that tears contain high levels of cystatin-like immunoreactive material, amounting to about 10% of tear proteins. In microgram cystatin S/mg protein the values were: right eye: 94.7 +/- 9.9; left eye: 115.5 +/- 14.8; n = 12. Cystatin levels of tears collected using capillary tubes were comparable: 120.7 +/- 19 micrograms/mg protein (n = 10). Immunoblots of tear fluids revealed a protein of about 14,000 molecular weight which reacted with antihuman cystatin SN monoclonal antibodies. Protein(s) of similar molecular weight were visualized using antibodies against human cystatins S and C. Less abundant additional cystatin-like immuno-reactive proteins were detected by using the two latter antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Transduction of proteins and other macromolecules constitutes a potent technology to analyze cell functions and to achieve therapeutic interventions. In general, fusion proteins with protein transduction domains, such as TAT, are produced in a bacterial expression system. Here we describe the generation of a mammalian expression vector coding for TAT-EGFP fusion protein. Transfection of CHO-K1 cells by this vector and subsequent selection by Zeocin resulted in cell lines that express and secrete EGFP, a variant of the green fluorescent protein GFP. The ultimate cell line was produced by first cloning the stable integrants and subsequent selection of EGFP-expressing cells by flow cytometric sorting. In the resulting cell line approximately 98% of cells express EGFP. Using the same methodology, we generated cell lines that express DsRed fluorescent protein. The advantages of using such a mammalian expression system include the ease of generating TAT fusion proteins and the potential for sustained production of such proteins in vitro and, potentially, 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 β-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. (J Histochem Cytochem 45:1533–1545, 1997)

Background: Cystatin C, a naturally occuring inhibitor of cysteine proteinases, belongs to family 2 of the cystatin superfamily. While cystatins in general, and cystatin C specifically, are expressed in various cell types and found in biological fluids, cystatins in ocular structures have not been investigated. In the present study, the expression of cystatin C mRNA in the eye of the rat was studied. Methods: Total RNA was extracted from eyes as well as from pooled corneae, retinas, lenses, sclerae, and corneae of adult rats. Cystatin C mRNA was detected in the RNA samples by reverse transcriptase—polymerase chain reaction and Northern blot hybridization. In addition, in situ hybridizations of formalin-fixed cryostat sections were carried out using a digoxigenin-labeled cystatin C probe. Results: Cystatin C mRNA was demonstrated in total RNAs extracted from the eye, sclera, and retina, but not in RNAs isolated from the cornea and lens. In situ hybridizations revealed cystatin C mRNA in most of the stromal cells of the sclera. In the retina, a strong signal was localized in the outer nuclear layer. The distribution of the reaction product suggested that in the retina Muller cells and rod cells are the primary sites of expression of cystatin C. In addition, some glial cells in the inner nuclear and ganglion cell layers were stained. No specific signal for cystatin C mRNA was detected in the cornea, lens, iris, ciliary body, and choroid. Conclusions: In the eye of the rat, significant levels of cystatin C mRNA are detected in the sclera and retina. In the sclera cystatin C may play a role in modulating the activities of cysteine proteinases, mostly cathepsins, involved in the turnover and remodeling of the stroma. In the retina, cystatins synthesized and presumably released by Muller cells and rod cells may have a protective function against the harmful effects of cysteine proteinases released under physiologic and pathologic conditions. © 1994 Wiley-Liss, Inc.

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.

Cystatins are widely distributed natural inhibitors of cysteine proteinase. They occur both intra and extracellularly in various cells and tissue fluids including tears. Using an immunofluorescence technique with antibodies against rat cystatin S, an inhibitor of submandibular gland origin, cystatin-like immunoreactive material was demonstrated in the acinar cells of the exorbital lacrimal gland of the rat. Administration of the cholinergic agonist carbachol caused a depletion of cystatin from the acinar cells. This depletion was followed by a partial restitution in 6-8 h. Administration of the beta-adrenergic agonist isoproterenol for 4 days, which caused a marked hypertrophy of the submandibular gland, had no effect on the structure, weight, or cystatin content of the exorbital lacrimal gland. After such treatment, however, single large cells with intense staining for cystatin were encountered. Cystatin-like immunoreactive material was also demonstrated in human lacrimal gland using antibodies against human cystatin S. These data suggest the notion that tear cystatins are secreted by the lacrimal glands.

The submandibular glands of mice with testicular feminization (Tfm/Y) and their normal adult male littermates (Ta/Y) were studied by immunocytochemical techniques for the demonstration of epidermal growth factor (EGF), nerve growth factor (NGF), renin and protease A. In the glands of both the affected and normal males, these polypeptides were restricted to cells of the granular convoluted tubules (GCT), with the exception of protease A, which was also found in small amounts in striated duct cells. Compared to those of Ta/Y males, GCTs were narrower in the glands of Tfm/Y mice and contained a markedly reduced number of cells immunoreactive for EGF, NGF and renin. However, the number of GCT cells that stained for protease A in the glands of Tfm/Y males was not as drastically decreased.

Epidermal growth factor (EGF) (urogastrone) is found in high concentrations in mouse and human milk. The origin of milk EGF is unknown. Milk samples were collected from lactating mice 2-6 h after the intravenous administration of a tracer dose of 125I-labeled EGF. The milk contained relatively high levels of radioactivity of which 35–46% was precipitated by trichloroacetic acid-phosphotungstic acid (TCA-PTA) and 26% by a specific antimouse EGF antiserum. Part of the radioactivity in milk was eluted from a Bio-Gel P-10 column at the point at which pure standard 125I-EGF was eluted. These data indicate that the mammary gland of the lactating mouse is capable of sequestering and transporting 125I-EGF into milk. Administration of a thousand-fold excess of unlabeled EGF caused no reduction in TCA-PTA-precipitable radioactivity in milk samples of mice given 125I-EGF. When mice were given 10 micrograms of unlabeled EGF and milk was collected 4 h later, compared with controls the EGF level in milk was doubled. Administration of EGF had no effect on lactose and protein concentrations in the milk, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed no qualitative or quantitative changes in the major milk proteins. Milk collected from lactating mice that were sialoadenectomized 6 mo earlier contained about 50% less EGF compared with controls. After the administration of 125I-EGF high concentrations of radioactivity were also found in the mammary and submandibular glands and in the stomach. In the latter organs, however, 95–96% of the radioactivity was in the acid-soluble fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

The nature of the trophic response of the mouse submandibular gland to thyroxine (T4) was examined. Adult female Swiss-Webster mice were given daily subcutaneous injections of T4 (1 microgram/gm body weight) for two or four days; two injections of tritiated thymidine (3H-TdR) were given 24 and 29 hours after the last injection of hormone, and the mice were killed one hour after the last injection of 3H-TdR. One gland was analyzed chemically for DNA content and for incorporation of 3H-TdR, while the other was used to prepare autoradiograms. The cellular composition of each gland was analyzed by counting 1000 nuclei, and the frequency and labelling index (LI) of six cell types were established. A rise in specific activity of DNA and a fall in its concentration were seen in response to T4. The LI for the entire gland more than doubled. The LIs and frequencies of granular convoluted tubule and granular intercalated duct cells were increased more than those of acinar and nongranular intercalated duct cells; striated and excretory duct cells were not affected. It is concluded that the enlargement of the submandibular gland of the mouse caused by T4 is due to both hyperplastic and hypertrophic effects.

The time of appearance and the pattern of localization of epidermal growth factor (EGF) in submandibular glands of mice was studied during postnatal development immunocytochemically. EGF was first detectable in the granular convoluted tubule (GCT) cells in the glands of males at 20 days of age and of females at 30 days of age. Development of GCT cells containing EGF was rapid in males, approaching adult conditions by 45 days of age. In females EGF- containing GCTs developed more slowly and irregularly, and did not reach adult status by 45 days of age. It is concluded that EGF is restricted during postnatal development to the GCT cells, and that these cells and the distribution of EGF are represented dimorphically from their first appearance in the submandibular glands of both sexes.

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.

The localization of renin in the developing mouse submandibular gland was studied immunocytochemically using the unlabelled antibody-enzyme method of Sternberger (1974). Bouin-fixed submandibular glands of mice of both sexes were examined at 5-day-intervals from birth (day 0) to 50 days of age. At all stages studied, only granular convoluted tubule (GCT) cells stained immunocytochemically for renin; such cells were first seen in glands of 30-day-old males and of 30-day-old females. The size and number of renin-containing GCT cells increased rapidly in males, attaining adult status by 50 days of age. In females, differentiation of GCT cells immunoreactive for renin was slower and less regular than in males, and at 50 days of age the GCT segment had not yet reached adult conditions with respect to the distribution of renin. Renin appears in GCT cells at later ages than other GCT cell products (e.g., EGF and amylase), suggesting the existence of independent developmental control for the expression of various biologically active substances in the GCTs.

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.

Chronic administration of the β-adrenergic agonist isoproterenol (IPR) leads to marked hyperplastic/hypertrophic enlargements of the parotid and submandibular glands in rats and mice with concomitant changes in the composition of both the glands and the saliva. Conspicuous among the alterations of the submandibular saliva is the appearance of a 13,000 Mr protein, termed LM (large mobile) protein. Repeated amputation of the lower incisor teeth also causes enlargements of the major salivary glands in rats. In this study, we have compared the enlargements of submandibular glands of rats produced by IPR administration or teeth amputation with respect to the relative levels of the LM protein in gland extracts and saliva. Adminstration of IPR-HCI (40 mg/kg) twice daily for 5 days or amputation of the lower incisor teeth 3 times a week for 3 weeks resulted in a 2.2-fold increase in the weight of the submandibular gland. Amputation for one week led to a 1.4-fold increase in gland weight. Double immunodiffusion in agar antibodies against the purified LM protein gave a single preciptin line with gland extracts and saliva of IPR-treated and teeth-amputated rats, indicating immunological identity of the reacting antigens. No precipitin lines were seen with gland extracts or saliva of untreated rats. Immunoblots of pooled saliva obtained from IPR-treated or teeth-amputated rats revealed a single protein band of the same electrophoretic mobility in SDS-polyacrylamide gels when stained using anti-LM antibodies. The relative concentrations of LM protein in gland extracts and saliva were measured by a solid-phase enzyme-linked immunoabsorption assay using antibodies aginast the purified LM protein. In gland extracts of untreated rats, LM protein was not measurable. Both amputation and adminstration of IPR resulted in marked increase in the relative concentrations of the LM protein. However, the relative concentration of LM protein in extracts of glands of teeth-amputated rats was only about one-fifth of that found in extracts of glands of IPR -treated animals irrespective of the duration of the amputation. LM protein was localized immunocytochemically in the cytoplasm of acinar cells and in the striated duct cells. These data indicate that both amputation of incisor teeth and β-agonist administration lead to the induction of the same protein in the submandibular glands of rats, although the mechanism by which these two types of interventions cause enlargement of the gland may not be identical.

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 [ 3 H]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 [ 3 H]-dihydroalprenolol compared with normal tissue. Catecholamine sensitive adenylate cyclase activity varies independently of the number of specific [ 3 H]dihydroalprenolol binding sites during isoproterenol-induced growth. Previously-described differences 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.

Administration of the beta-adrenergic drug isoproterenol (IPR) produces hyperplastic and hypertrophic enlargements of the submandibular gland of the rat and induces the synthesis of specific proteins in this organ. One of these proteins, the LM (large mobile) protein, was demonstrated immunocytochemically in the submandibular glands of developing untreated and IPR-treated rats. Immunoreactive LM protein was absent in the glands of 20-day-old fetuses and 1- and 2-day-old rats. It was localized in the proacinar and immature acinar cells in the glands of 6- to 21-day-old animals, but it was undetectable at 28 days of age. In the glands of adult rats, secretory granules of the granular convoluted tubule cells showed immunostaining for the LM protein which was also present in trace amounts in the acinar cells. Daily administration of IPR for 5 days to newborn or 8- or 15-day-old rats caused an apparent acceleration of proacinar/acinar cell differentiation, and consequently it increased the frequency of cells immunostained for the LM protein as well as the amount of immunoreactive material in these cells. Thus, the expression of LM protein in the submandibular gland is developmentally regulated, and it is restricted to the stage of differentiation of proacinar cells from terminal tubule cells. IPR is capable of inducing this protein in fully differentiated acinar cells in 3-week-old or older animals.

Epidermal growth factor (EGF), and androgen-dependent polypeptide, occurs in high concentration in male mouse submandibular gland. Glands of adult male and female mice of six inbred strains (129/J, C57BL/6J, C58/J, SWR/J, RF/J, A/J) were assayed for EGF by radioimmunoassay. In all strains, the glands of males contained 30 to 500-fold more EGF than those of females. Furthermore, significant differences in EGF content were found among the various strains in both sexes, the highest amount of EGF was present in RF/J and the lowest in C57BL/6J, with a ratio of three in the males and four in the females of the two strains, respectively. Factors that effect EGF levels were analyzed further, using these two strains. EGF was measurable in the glands of mice of both strains at 21 days of age and increased rapidly thereafter, up to 14 weeks of age. Throughout postnatal development, the level of EGF was greater in the glands of RF/J mice than in those of the C57BL/6J animals. Thirty days after castration, the EGF levels were reduced by about 98% in both strains, but the strain difference was not abolished. Testosterone implants (1 mg in Silastic tube) in castrated mice induced EGF levels six- to ten-fold compared to castrates. Even in induced animals, which had similar plasma testosterone levels, as measured by radioimmunoassays, the difference in EGF levels between the two strains was manifest. Such a difference, however, was not seen after the daily administration of 5-alpha-dihydrotestosterone for 3-14 days. Immunocytochemical straining for EGF also indicated a higher concentration of the polypeptide in the glands of RF/J mice than in those of C57BL/6J animals, and confirmed the exclusive localization of EGF in the cells of the granular convoluted tubules (GCT). According to our morphometric analysis, in the glands of male RF/J mice the GCT compartment occupied a greater portion (8% greater, P less than 0.001) of the gland volume than in C57BL/6J mice. The difference in the relative GCT volumes in the glands of female mice of the two strains was, however, statistically not significant. There was no direct correlation between the amount of EGF and the relative volume of the GCTs in the two strains. The evidence obtained implies that strain difference in submandibular-gland EGF levels are determined genetically.

Radioimmunoassays and immunocytochemical techniques were used to assess the effect of cyclocytidine, an antitumor agent, on the level and localization of Epidermal Growth Factor (EGF) in the submandibular gland of the male mouse. A single intraperitoneal injection of 150 mg/kg of cyclocytidine caused, within 6 h, a degranulation of the granular convoluted tubule (GCT) cells and reduced the concentration of immunoreactive EGF in gland extracts by more than 90 %. This effect was largely abolished by the administration of dibenzyline but not by propranolol, indicating that the secretory effect of the drug on the GCT cells is mediated by α-adrenergic receptors. By immunocytochemical staining EGF was localized to the GCT cells. Immunocytochemical staining revealed the same trends in changes in EGF concentration as the radioimmunoassays. However, even at the peak of the cyclocytidine effect there were cells which retained their secretory granules and apparently their EGF complement. In addition, there was a lobular variation in the secretory response. Cyclocytidine caused a transient increase in the blood level of EGF. Furthermore, it stimulated amylase secretion from the gland, which also involved α-adrenergic receptors. Cyclocytidine will be useful in future analyses of the release of various biologically active substances from the GCT cells of the mouse submandibular gland.

The effects of 5-bromodeoxyuridine (BrdU) on the postnatal differentiation of rat submandibular gland and on the isoproterenol-induced changes of differentiation were studied. The rats were injected with BrdU for three consecutive days, beginning at two days of age. The total dose of BrdU was 0.9 mg/g body weight. BrdU caused a severe retardation of growth up to 15 days of age. A rapid growth of the animals between 15 and 22 days indicated a recovery from the effect of BrdU. The growth of the submandibular gland was retarded similarly with a corresponding decrease in DNA, RNA and protein content. Incorporation of tritiated thymidine into the submandibular gland was not altered in the BrdU-treated animals at one and three days after the last injection of the analog. At days 15 and 22 the rate of thymidine incorporation was greater in the submandibular gland of BrdU-treated rats as compared to littermate controls. Isoproterenol stimulated thymidine incorporation into the submandibular glands of two-week-old rats. This stimulation was not observed in rats which received BrdU at age 7–9 days, prior to the administration of isoproterenol. Electron microscopic observations, including a quantitative analysis of the frequency distribution of the various cell types in the terminal tubules and developing acini, indicated a retardation of acinar cell differentiation in the glands of BrdU-treated rats. In addition, there was an increase in the number and size of the secretory granules in the terminal tubule cells. BrdU treatment, however, caused no obvious pathologic alterations in the submandibular gland. Administration of isoproterenol for five days, beginning at five days of age, caused an apparent acceleration of the differentiation of acinar cells. In the glands of isoproterenol-treated rats the acinar cells were enlarged and were filled with homogenous secretory granules. Pretreatment with BrdU partially inhibited the effects of isoproterenol on differentiation and hypertrophy of the submandibular gland. With the given dose of BrdU, approximately 5% of thymine was replaced by bromouracil in the DNA of the submandibular gland. Such a replacement would be consistent with a direct effect of BrdU on the cytodifferentiation in the submandibular gland. However, because of the severe retardation of growth of the BrdU-treated rats, indirect effects can not be excluded.

SummaryLiver cell injury of subacute ethionine intoxication of the rat is accentuated by rising ethionine levels in the diet. Methionine supplements have an increasingly inhibitory effect depending on concentration. That effects of even high levels of ethionine may be almost completely eliminated by high methionine levels speaks for the role of ethionine as a metabolic competitor of methionine rather than as a primary toxic agent. Methionine inhibits ductular cell proliferation much more effectively than the injury to hepatic cells, thus dissociating both lesions. Ductular cell proliferation and fiber formation run parallel.The authors are very grateful to Helene Tzitsikas and Roy Dube, Kenneth Thompson, and Jim Matthias for technical assistance.

A simple method is described which makes it possible to determine the relative amount of Schiff reagent bound to periodic acid oxidized tissue sections. It is based on the application of S35-labeled Schiff reagent. It was found that paraffin embedded tissue sections bind significant amounts of Schiff reagent without any previous treatment, but using appropriate control sections it is possible to determine the relative amounts of aldehyde groups created by periodic acid oxidation as opposed to the amounts in the controls.

The prenatal development of endogenous peroxidase activity in the submandibular gland of rat was investigated by means of the diaminobenzidine-H2O2 histochemical method. The submandibular gland of a 16-day-old fetus was composed of cords of uniform, undifferentiated cells which contained no secretory granules and revealed no peroxidase activity. Peroxidase activity first appeared at the 17th day of gestation in the cisternae of the rough endoplasmic reticulum and nuclear envelope in a few cells. At the 18th day of gestation cells which exhibited reaction products in the rough endoplasmic reticulum and nuclear envelope also contained secretory granules with a strong peroxidase activity. During the last days of gestation the number of peroxidase positive cells, which contained numerous secretory granules, increased. The peroxidase-containing cells are the immediate precursors of the proacinar cells of early postnatal stages. During the same time period, when the peroxidase-containing cells differentiated, a second cell type also differentiated in the cellular cords. The development of this cell type was marked by the appearance of secretory granules stainable with toluidine blue. Through the prenatal development, this cell type revealed no peroxidase activity and was identified with the terminal tubule cell of the newborn. The morphologic and cytochemical findings indicate that terminal tubule cells and proacinar cells are committed cells; the former differentiate toward 2nd order intercalated duct cells and the latter transform to mature acinar cells.

The post-natal growth of rat submandibular gland and the effect of isoproterenol on this process were studied. Between 2 and 42 days of age the DNA content of the gland increased linearly but the increase in RNA and protein content was more rapid after 29 days of age. The RNA: DNA and protein: DNA ratio increased linearly with age. The proliferative activity, measured by the incorporation of tritiated thymidine, was maximum in the gland of 7-day-old rats. It declined steadily to a low level in 42-day-old rats. A single injection of isoproterenol had no effect on thymidine incorporation in 2-day-old rats. The drug, however, stimulated DNA synthesis in older animals and the degree of stimulation was inversely correlated with the proliferative activity in control rats. Small doses of isoproterenol given to rats for 4 days between 2 and 5 days of age produced a hypertrophy of the submandibular gland. The same treatment between 7 and 10 days of age caused both hypertrophy and hyperplasia of the gland. It is concluded that both the regulation of growth and the regulation of induced cell proliferation are a function of cellular differentiation and that cell proliferation can be induced only in cells that reached a certain degree of differentiation.

Adenylate cyclase activity was measured in homogenates of submandibular glands of 1 to 42 day old rats. During this period of time the gland reached its final stage of differentiation. Adenylate cyclase activity was higher in the glands of one day old rats than in those of 7 and 14 day old animals. Between 14 and 28 days of age the enzyme activity more than doubled and approached the level that characterized the glands of adult animals. Fluoride (10mM) stimulated the enzyme activity in all age groups but the stimulation was less in the case of one day old rats as compared to older animals. Isoproterenol (10 −4 M) stimulated adenylate cyclase by 50–60% in the gland of adult rats but had no effect on the enzyme activity in 7 to 28 day old animals. Administration of isoproterenol for 5 days to 9 day old rats increased the weight of the submandibular gland by 70 per cent. Total adenylate cyclase activity increased parallel with the weight of the gland but the specific activity of the enzyme remained unchanged. It is concluded that during the postnatal development of the submandibular gland the rapid increase in adenylate cyclase activity occurs after weaning and it coincides with an accelerated rate of functional differentiation of the acinar cells.

The localization of peroxidase activity was studied in the developing submandibular gland of normal and isoproterenol-treated rats using a diaminobenzidine-H2O2 method. During postnatal development peroxidase activity was localized in the proacinar and acinar cells. The proacinar cells were characterized by the presence of polymorphic secretory granules that gave a strong peroxidase reaction, particularly in the glands of 1-day-old rats. In addition to the secretory granules, enzyme activity was demonstrated in the rough endoplasmic reticulum and nuclear envelope. The terminal tubule cells and duct cells were devoid of peroxidase activity. The secretory granules in the fully developed acinar cells revealed little or no enzyme activity. Isoproterenol stimulated the secretion of the peroxidase-positive granules from the proacinar cells. The stimulation was followed by a reaccumulation of peroxidase-positive secretory material. During this process enzyme activity was demonstrable in the Golgi complex. Isoproterenol had no effect on the terminal tubule cells. A less effective depletion of the secretory granules from the proacinar cells was seen after pilocarpine administration. Chronic administration of isoproterenol to 5-day-old rats led to an acceleration of the differentiation of acinar cells and to a hypertrophy of the gland, without significant change in the localization of peroxidase activity.

The technique of Polyacrylamide gel electrophoresis for acid phosphatases is described. This permits the separation of the soluble acid phosphatases of rat liver into three fractions.

The incorporation of 14 C-leucine into the soluble proteins of the submandibular gland was studied in rats of different ages. The proteins were separated by ion-exchange chromatography and by electrophoresis on polyacrylamide gels. The pattern of protein synthesis during postnatal development undergoes a considerable change. Certain secretory proteins are not synthesized before 3 wk of age. Isoproterenol, given for 5 days beginning at 6 days of age, accelerates the postnatal differentiation of the submandibular gland. This is manifested not only in a morphological differentiation of acinar cells but in the induction of the synthesis of proteins characteristic for more advanced stages of development.

Summary The effect of the administration of isoproterenol (IPR) on the uptake and steady state distribution of 14 C-amino-isobutyric acid ( 14 C-AIB) and 14 C-cycloleucine was measured in rats. Isoproterenol in small doses specifically enhanced the uptake of 14 C-AIB and 14 C-cycloleucine by the submandibular gland. In large doses the drug increased the uptake of the amino acid analogues also by the liver and diaphragmatic muscle. Removal of the superior cervical ganglion reduced the uptake of 14 C-AIB by the submandibular gland on the operated side. It is suggested that regulation of amino acid transport is one of the mechanisms by which IPR effects the growth of the submandibular gland.

Conventional light microscopic observation of the mesenchymal cells and of the sinusoidal wall in various types of human liver injury were supplemented by histochemical, electron microscopic, and immunocytochemical investigations. The cells were subdivided into endothelial, phagocytic, protein-forming, fibroblastic, and hematic cells on the basis of structural characteristics. Although transitions between endothelial and phagocytic cells, as well as between protein-forming and phagocytic cells, are suggested, the division presented does not necessarily imply postembryonal derivation from a single precursor reticulum cell but could also be explained by different cell lines proliferating as a result of stimulation. The same types of cells are found in the parenchyma around hepatic necrosis and granulomas as well as in portal tracts and in septa of cirrhosis. The criteria used for phagocytosis were lipofuscin pigment, PAS reaction, acid phosphatase reaction, and phagosomes. For protein formation the criteria were gamma globulin localization and abundant endoplasmic reticulum. For fibroplasia they were electron microscopically characteristic filaments becoming fibers extracellularly. Acute liver cell injury is characterized by proliferation of endothelial and phagocytic cells to which are added protein-forming and fibroblastic cells in chronic injury. Hypergammaglobulinemia and erratic immunologic reactions in cirrhosis were related to activation of the hepatic mesenchyma in chronic liver cell injury. In liver cell injury the tissue space is widened and exhibits accentuated PAS, ATPase, and alkaline phosphatase reaction. Formation of excess fibers in cirrhosis and peripheral “piecemeal” necrosis is eventually followed by formation of a basement membrane in the hepatic sinusoids closing the previously open circulation. The reduced permeability of the sinusoidal wall resulting from an increase in cells and a fibrous barrier with a basement membrane, together with alterations of the hepatocellular microvilli, increases hepatic insufficiency.

The observation of Holt that the storage of formalin fixed tissues in sucrose-gum acacia solution preserves acid phosphatase activity was confirmed. The same was found for 5-nucleotidase. Adenosinetriphosphatase activity is present after storage both in formalin and in sucrose-gum acacia solution. However, the staining for acid phosphatase activity after preservation in sucrose and for adenosinetriphosphatase activity after preservation in formalin and in sucrose solution revealed artefacts decreasing the usefulness of this procedure for routine application.

Summary Acid phosphatase, five-nucleotidase, ATPase, aminopeptidase, alkaline phosphatase and non-specific esterase activities were demonstrated in the schistosomal ova in the livers of mice experimentally infected with Schistosoma mansoni. In addition, the ovum contained a complex carbohydrate which is probably its main antigenic component. Granulomatous tissue around the ova displayed histochemical reactions characteristic of chronic proliferative inflammation. The reticuloendothelial cells in liver and spleen exhibited high acid phosphatase activity.

Administration of isoproterenol (IPR) greatly reduced the acid and alkaline ribonuclease activities in rat parotid and submandibular glands in acute experiments. RNase activity was decreased by about 70 % in parotid gland enlarged by the administration of isoproterenol for 7 days. On the other hand, the hypertrophic and hyperplastic submandibular gland displayed higher RNase activities as compared with control.

A single injection of isoproterenol greatly stimulates DNA synthesis in the submandibular glands of rat and mouse. With the administration of cell-free homogenates of salivary glands this stimulatory effect can be suppressed. Homogenates of salivary glands of rats or mice treated with the drug, however, have no such effect. An active factor, which suppresses the isoproterenol-stimulated DNA synthesis has been partially purified from mouse submandibular gland.

Serum-free culture conditions that permit the continuous growth of A-431 human epidermoid carcinoma cells were developed. In Dulbecco's modified Eagle's synthetic nutritional medium (DME) supplemented with fetuin, insulin, transferrin, biotin, and oleic acid-fatty acid-free bovine serum albumin complex A-431 cells grew at a rate comparable to that observed in the presence of calf or fetal calf serum. Of the factors tested, oleic acid had the most pronounced stimulatory effect on the growth and [3H]-thymidine incorporation of A-431 cells in serum-free medium. A-431 cells have a high number of receptors for epidermal growth factor (EGF); they bind and rapidly internalize EGF. Nevertheless, EGF did not stimulate either the growth or the [3H]-thymidine incorporation of these cells. Analyses of [125I]-EGF binding data indicated that A-431 cells grown in the presence of calf serum had about 3.2-3.9 X 10(6) specific, saturable EGF receptor sites on their surface. Linear Scatchard plots indicated a single class of noninteracting receptors with an apparent equilibrium dissociation constant of about 2.8 X 10(-9) M. The average number of receptors of A-431 cells maintained in DME supplemented with only fetuin, insulin, and transferrin for several months was significantly less, 1.54 X 10(6), than that of A-431 stock cells cultured in the same medium for 2 days only (2.68 X 10(6)). The apparent dissociation constants for the same cell populations were, however, similar, 4.5 X 10(-9) M and 4.1 X 10(-9) M, respectively. Stimulation of growth by oleic acid resulted in about 20% decrease in the average number of receptor sites, with an increase in the apparent equilibrium dissociation constant.