David J. Culp

Light and electron microscopic immunolocalization of rat submandibular gland mucin glycoprotein and glutamine/glutamic acid-rich proteins.

We studied the subcellular localization of two major secretory products of adult rat submandibular gland (RSMG), blood group A-reactive mucin glycoprotein and glutamine/glutamic acid-rich protein (GRP), by light and electron microscopic immunocytochemistry. The structure of the major neutral oligosaccharide of the mucin was shown to be: GalNAc alpha 1,3(Fuc alpha 1,2)Gal beta 1,3GalNAc. A mouse monoclonal antibody (1F9) with specificity for blood group A determinants was prepared against the mucin. The antibody recognized a single band of approximately 114 KD on Western blots of RSMG extract. A previously characterized monoclonal antibody (59) against GRP (Mirels et al.: J Biol Chem 262: 7289, 1987) reacted with a doublet of 45-50 KD on Western blots of extraparotid saliva. Immunofluorescence and immunoperoxidase staining of cryostat sections of RSMG with anti-mucin antibodies and anti-GRP antibodies revealed reactivity in acinar cells of the gland. No specific labeling was seen in duct cells of RSMG or in mucous acinar cells of the adjacent sublingual gland. Post-embedding immunogold labeling of thin sections of glutaraldehyde-fixed RSMG with anti-mucin showed strong labeling of the Golgi apparatus and secretory granules of acinar cells. Gold particles were seen mainly over electron-lucent areas of the granules. No labeling occurred over the endoplasmic reticulum. The labeling pattern with the anti-GRP antibodies was similar, except that both electron-dense and -lucent areas of the granules were labeled, and the endoplasmic reticulum was reactive. Double labeling with two different sizes of gold particles showed that both mucin and GRP co-localized in the same granules. Pre-absorption of the antibodies with their respective antigens eliminated immunolabeling of the acinar cells. These antibodies will be useful in studies of cell differentiation in RSMG and of synthesis, processing, and packaging of RSMG secretory products.

The Effect of Chronic Atropine Treatment on Salivary Composition and Caries in Rats

Many instances of salivary dysfunction in humans can be traced to the use of medications that have hyposalivary side-effects. In this study, atropine, a cholinergic antagonist, was administered chronically to rats by use of osmotic mini-pumps. Steady-state blood levels, similar to levels obtained in human multiple oral dosing, were thus maintained. Atropine delivered in this manner for 24 days was found to decrease protein concentration of parotid saliva (p<0.05) elicited by pilocarpine, and to increase smooth-surface caries scores (p<0.05) in rats fed a cariogenic diet. Parotid saliva collected via ductal cannulation from rats subjected to chronic atropine administration (and stimulated to secrete by pilocarpine) exhibited increased levels of two basic proline-rich proteins (Peak A and SP-3), as evaluated by SDS-PAGE, compared with those observed in saliva from controls. Cannulation of sublingual glands in animals receiving high doses of atropine produced no measurable secretion upon pilocarpine stimulation. Carbachol stimulation of dispersed cell aggregates of sublingual glands from sham-operated and high-dose atropine groups indicated that the glands responded similarly once the antagonist was washed from the system, implying that the lack of secretion in vivo was caused by antagonism of the cholinergic receptor by atropine. Our observations suggest that this model system can be exploited for determination of the effects of chronic administration of hyposalivary drugs on salivary composition and caries rates.

Biochemical and immunological studies and assay of rat sublingual mucins.

Original studies of rat sublingual mucins raised questions as to the existence of a second mucin species as distinguished by binding to hydroxyapatite. The existence of multiple mucin species is of concern in pharmacological studies of mucous-cell secretion as each species could represent distinct mucous-cell populations that respond differently to secretagogues. Thus a separate hydroxyapatite-bound mucin pool expressed in rat sublingual glands was isolated and characterized. Biochemical comparison of hydroxyapatite-bound mucins to total and hydroxyapatite-unbound sublingual mucins demonstrated no substantial differences in either amino acid and carbohydrate contents or in size distributions. In addition, a radioimmunoassay was developed using antisera prepared previously against unbound mucins. The three mucin pools exhibited equal specificities in displacement of radiolabelled unbound mucin tracer in the radioimmunoassay. Thus, bound and unbound mucins are indistinguishable, both immunologically and in biochemical composition. The radioimmunoassay was then evaluated for use in pharmacological studies of acinar mucous-cell secretion. Measurement by radioimmunoassay of secretion from isolated acini in response to carbachol was concentration-dependent (EC50 approx. 0.3 microM and maximal stimulation at 1 microM carbachol). In immunolocalization studies the antiserum was highly selective for mucous cells, recognized all mucous cells within histological sections, and was localized subcellularly to mucous-cell secretion granules and trans-Golgi, further validating the radioimmunoassay as a method to detect exocrine secretion from the entire pool of acinar mucous cells. Moreover, the radioimmunoassay was compared and found equivalent to an acid-precipitation method to assess relative secretion, suggesting the acid-precipitation method is also valid for pharmacological studies of isolated acini.

Variable Effects of Soman on Macromolecular Secretion by Ferret Trachea

The purpose of this study was to examine the effect of the anticholinesterase agent, soman, on macromolecular secretion by ferret trachea, in vitro. We mounted pieces of ferret trachea in Ussing-type chambers. Secreted sulfated macromolecules were radiolabeled by adding 500 microCi of 35SO4 to the submucosal medium and incubating for 17 hr. Soman added to the submucosal side produced a concentration-dependent increase in radiolabeled macromolecular release with a maximal secretory response (mean +/- SD) of 202 +/- 125% (n = 8) relative to the basal secretion rate at a concentration of 10(-7) M. The addition of either 10(-6) M pralidoxime (acetylcholinesterase reactivator) or 10(-6) M atropine blocked the response to 10(-7) M soman. At soman concentrations greater than 10(-7) M, secretion rate decreased and was not significantly different from basal secretion. Additional experiments utilizing acetylcholine and the acetylcholinesterase inhibitor, physostigmine, suggest that inhibition of secretion by high concentrations of soman may be due to a secondary antagonistic effect of soman on muscarinic receptors.

Cell lines expressing the adenovirus e1a 12s protein derived from rat sublingual glands

Dear Editor: Mucous cells are highly regulated exocrine cells that synthesize and secrete high-molecular weight mucin glycoproteins, the major secretory component of salivary mucous glands such as the major sublingual and many of the minor salivary glands (1). Currently, the absence of well-differentiated salivary mucous cell lines has hindered the study of mechanisms regulating mucous cell-specific gene expression and exocrine secretion. Recently, He et al. (8) used a retroviral vector system encoding for the adenovirus E1A 12S protein to establish immortalized cells from seromucous salivary glands, rat submandibular glands. They derived a clonal cell line, 2C-2, which increased cellular cAMP levels in response to ]3-adrenergic agonist, and synthesized blood group A reactive oligosaccharides, similar to those present in the low-molecular weight mucin synthesized by rat submandibular glands. Therefore, we tested whether this retroviral vector system would immortalize rat sublingual eells and also provide cell lines expressing the mucous cell phenotype. We first prepared isolated cells by collagenase/dispase digestion [2.5 mg/nd in phosphate-buffered saline (PBS) for 30 rain at 37 ~ C] of sublingual glands excised from eight Wistar rats (28 d old). Cytospins were subjected to immunocytochemistry using an indirect peroxidase method described previously (3) to determine the presence of mucous cells [antisublingual mucin antisera (10)] and serous demilune cells or striated ductal cells [anti-B1 antibody (10)]. As shown in Fig. 1, about half the cells stained either strongly or lightly for mucin (panelA), whereas about 15% of the cells displayed diffuse staining for anti-B1 antibody (panel B). Unstained cells may represent either degranulated acinar cells, myoepithelial cells, intercalated ductal cells, or fibroblasts. Isolated cells were cultured and subsequently infected with E1A 12S retroviral vector (kindly provided by Dr. L. A. Tabak) as described by He et al. (8). After selection with G418 and cloning by limited dilution, we derived eight cell lines: AD-6, AE-5, BC-4, DD3, HD-6, HE-4, HE-5, and JD-6. All cell lines, as well as the original parent cells used to derive clonal cell lines, were epithelioid in appearance by phase-contrast microscopy (see Fig. 1 C and D). Cells expressed the adenovirus E1A 12S protein, as determined in immunoprecipitation experiments (not shown) using a monoelonal antibody to adenovirus EIA antigens (Oncogene Science, Manhasset, NY). Cytospins prepared from each cell line (Passage 6) and of parent cells were probed for expression of cytokeratins and for markers specific for different epithelial cell types in rat sublingual glands. All cell lines, as well as the parent cells, expressed cytokeratins but were negative for antisublingual mucin and anti-B1 reactivity. When stained with Alcian blue-periodic acid Schiff (AB/PAS), none of the cell lines or parent cells exhibited positively stained cytoplasmic granules that would suggest the phenotypic expression of a differ-