Ultrastructural immunocytochemical localization of secretory proteins in autophagic vacuoles of parotid acinar cells of starved rats

Previous studies have shown that reduction of mastication has marked effects on the structure and biochemistry of the rat parotid gland. Acute starvation results in the formation in the acinar cells of large autophagic vacuoles which contain lysosomal hydrolases and within which secretory granules appear to undergo degradation. In this study we used electron microscopic immunocytochemistry and antibodies to two secretory proteins, alpha-amylase and B1-immunoreactive protein, to determine whether secretory proteins are present in autophagic vacuoles of parotid acinar cells of starved rats. Small vacuoles were observed after 24-h starvation; they increased in size and number up to 72-h starvation. Both secretory proteins were present in the secretory granules and in the dense content of the autophagic vacuoles, as shown by immunogold labelling. The lighter matrix of the vacuoles was unlabelled. These findings confirm that secretory granules may fuse with lysosomal structures, where their content of secretory proteins is presumably degraded. Thus, the rat parotid appears to be similar to other secretory cells in which cellular levels of stored secretory proteins may be regulated by the process of crinophagy.     

Interaction of SNARE proteins in rat parotid acinar cells

The protein-protein interaction between [soluble NSF attachment protein (SNAP) receptor] (SNARE) proteins found in the lysate of parotid acinar cells was investigated. Immunoblotting analysis showed that parotid acini contain both syntaxin-4 and SNAP-23, plausible candidates of target membranes (t-) SNAREs in non-neuronal cells. However, when vesicle-associated membrane protein (VAMP)-2 was immunoprecipitated from lysates of parotid acinar cells, syntaxin-4 and SNAP-23 were not coprecipitated with VAMP-2, although syntaxin-1 and SNAP-25, t-SNAREs in neuronal cells, were clearly coprecipitated with VAMP-2 from brain lysates. Inversely, when syntaxin-4 was immunoprecipitated from parotid lysates, SNAP-23, Munc18c, and N-ethylmaleimide-sensitive fusion protein (NSF) were coprecipitated, but VAMP-2 was again undetectable. When proteins in the crude secretory-granule fraction were biotinylated and then immunoprecipitated with anti-VAMP-2, 35- and 80-kDa proteins were coprecipitated along with VAMP-2. These results suggest that the interaction between syntaxin-4, SNAP-23 and VAMP-2 is fairly weak and their concentrations in the cell lysate are insufficient to make a readily detectable complex, and that bindings between these proteins are hindered by other proteins in parotid acinar cells.     

Ultrastructural changes induced by actinomycin D in acinar cells in submandibular glands of rats

Actinomycin D is a cytotoxic antibiotic which suppresses RNA and protein synthesis in many cells with consequential changes in the morphology and arrangement of organelles. Its effects on the fine structure of acinar cells in submandibular glands were studied in adult rats which had each received 75 μg of actinomycin D in a single intraperitoneal injection. There were no detectable structural changes in the first 24 hr. Three and 6 days following the injections, the arrangement and shape of the cells and the location and morphology of some cytoplasmic structures and organelles were aberrant. Ballooning degeneration in many mitochondria and development of numerous vesicles, cytolysosomes and myelin figures were prominent features. By the 9th day, there was evidence of early resumption of synthesis and secretion granules were increasing in numbers. Whorled arrangements of endoplasmic reticulum were distinctive in the treated cells.     

Calcium and cGMP in isolated rat parotid acinar cells

The role of calcium ions in the activation of guanylate cyclase and in the propagation of the effects of cGMP during activation of the muscarinic cholinergic receptors was studied in isolated rat parotid acinar cells. It was demonstrated that the requirement for extracellular calcium is complete in the transduction of information from the muscarinic receptor to the guanylate cyclase, and partial in the steps beyond the synthesis of cGMP.     

Isoproterenol-induced changes in cell cycle kinetics of parotid gland acinar cells in 8-day-old rats.

Isoproterenol (IPR) was shown to alter some parameters of the parotid gland acinar cell cycle in 8-year-old rats. Although the deoxyribonucleic acid (DNA) synthetic period (S) was decreased and the pre-DNA synthetic (G1) period was lengthened, there were no differences in the total cycle time. There was an increase in the 3H-thymidine labeling index from 35.7 to 50.8% combined with a decrease in the value of the mitotic index from 2.64 to 2.16% after IPR was administered. The results suggest that noncycling (G0) cells may be involved in the normal differentiation of the parotid gland and that IPR alters the transition of acinar cells between the cycling and noncycling state.     

Characterization of autonomic receptors in isolated human parotid acinar cells

Isolated human parotid acinar cells have been used for the in vitro characterization of the muscarinic cholinergic and alpha- and beta-adrenergic receptors of these cells. The agonist-antagonist interactions at the receptor level were studied, and the role of the receptor-activated cellular systems in the process of secretion was characterized.     

Lorazepam induces acinar cells apoptosis of rat parotid glands

To evaluate the apoptotic action of Lorazepam on acinar cells of the parotid glands of rats, forty male Wistar rats were separated into four groups. Saline was given to the control groups for thirty (GS30) and sixty days (GS60). L30 received Lorazepam for thirty days and GL30 + S30 Lorazepam for thirty days, and from the thirty-first day, saline. For the counting of apoptotic cell nuclei, TUNEL technique was applied. The statistical tests used were ANOVA and Tukey HSD. On the thirtieth day, there was no statistically significant difference between GL30 and GS30, in relation to the number of apoptotic cell nuclei and cell proliferation. There was a statistically significant difference in the number of apoptotic cell nuclei in GL30 compared to GS30. The percentage of apoptotic cell nuclei from the GL30+S30 was significantly higher in relation to GS60. A statistically significant reduction in the value of acinar cell nuclei was also observed between these groups. No significant difference was found in the values of cell proliferation among the GL30+S30 and GS60 groups. Lorazepam caused significant decrease in the number of acinar cell nuclei and larger percentage of cellular apoptotic nuclei in rat parotid glands.     

Ultrastructural morphology of secretory granules of submandibular and parotid salivary glands of man

Morphological variation among secretory granules of the same type of cell was found in the acini, intercalary and striated ducts, and collecting-ducts, and was greatest in serous and intercalary-duct cells. Secretory granules of monopartite and bipartite structure were seen in all these cell types; tripartite forms were seen in serous and intercalary-duct cells. A possible explanation for the variation is that one type of cell may be able to produce a range of secretory products and package them variously into secretory granules, thus creating different appearances.     

Intracellular localisation of SNARE proteins in rat parotid acinar cells: SNARE complexes on the apical plasma membrane

Intracellular localisation of soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptors (SNAREs) is an important factor in clarifying whether SNAREs regulate exocytosis in salivary glands. We investigated intracellular localisation of syntaxins 2, 3 and 4 and SNAP-23, which are thought to be target membrane (t)-SNAREs, in rat parotid gland by Western blotting and immunocytochemistry. Syntaxins 2 and 3 were localised in the apical plasma membrane (APM), and syntaxin 4 was localised in the plasma membrane. SNAP-23 was localised in the APM and intracellular membrane (ICM). In a yeast two-hybrid assay, syntaxins 2, 3 and 4 interacted with SNAP-23 and VAMP-3. Using immunoprecipitation methods, syntaxins 3 and 4 were seen to interact with VAMP-8 and SNAP-23 at the APM, respectively. SNAP-23 interacted with syntaxin 3, syntaxin 4, VAMP-2, VAMP-3 and VAMP-8. Many SNARE complexes were detected under non-stimulated/basic conditions in the parotid APM. Some of these complexes may have a role in exocytosis from parotid acinar cells.     

Ultrastructural morphology of secretory granules in pleomorphic adenoma of human parotid and submandibular salivary glands

Secretory granules were found in some of the cells lining the lumina of typical epithelial structures in pleomorphic adenomas. They were small, of varied electron density, and were mostly unipartite, sometimes bipartite and occasionally tripartite. They most closely resembled secretory granules of intercalary ductal cells of normal salivary glands.     

The uptake of sugars by isolated rat parotid acinar cells.

This study presents the basic transport mechanisms of the uptake of D-glucose and 3-0-methyl-D-glucose by isolated rat parotid acinar cells. The transport characteristics were studied in vitro using 14C-labeled isotopic analogues of these sugars.     

Morphological and functional characterization of isolated human parotid acinar cells.

A method for the isolation of human parotid acinar cells using enzymatic dispersion and milk mechanical forces has been developed. The isolated acinar cells are morphologically and functionally intact, thus suitable for studies of the cellular physiology of secretion.     

The actin-specific reagent jasplakinolide induces apoptosis in primary rat parotid acinar cells

Jasplakinolide is a reagent that stabilizes and polymerizes actin filaments and is a commonly used tool in cell biology. In primary rat parotid acinar cells, jasplakinolide partially inhibited the release of amylase induced by β-adrenergic receptor activation, as previously reported. However, in confocal microscopic observation with fluorescence conjugated anti-actin antibody, the jasplakinolide-treated cells not only showed decreased fluorescence intensity and aggregation of cortical F-actin but also revealed events characteristic of apoptosis such as cell shrinkage, membrane blebbing and apoptotic body formation. Such characteristic events of apoptosis were confirmed by transmission electron microscopy. The occurrence of apoptosis in jasplakinolide-treated cells was further confirmed by biochemical analysis: a DNA ladder was detected by electrophoresis, and DNA fragmentation was revealed using ELISA with an antibody to single-stranded DNA. Moreover, the degradation of fodrin was detected in jasplakinolide-treated cells by Western blotting, and the K(+) release induced by the fluid secretagogue carbachol was impaired. Taken together, these results demonstrate that jasplakinolide induces apoptosis and suppresses the secretory functions of rat parotid acinar cells.     

36Cl- and 86Rb+ uptake in rat parotid acinar cells

36Cl- uptake was markedly (85 per cent) inhibited by the loop diuretics, furosemide and bumetanide. Partial replacement of Na+ in the incubation medium (from 137 to 5 mM) reduced 36Cl- uptake 30 per cent; total replacement reduced uptake to 40-45 per cent of control values. Partial replacement of K+ (from 5.8 to 1 mM) decreased 36Cl- uptake approx. 45 per cent; complete replacement resulted in minimal levels of 36Cl- uptake (less than 10 per cent of controls). Ouabain reduced 36Cl- uptake approx. 55 per cent in the absence of bumetanide, but was without effect in its presence. 86Rb+ uptake was reduced approx. 85 per cent with bumetanide; complete replacement of Cl- with I- or gluconate-, decreased 86Rb+ uptake by 55 or 40 per cent respectively. The results support the notion that the bulk of Cl- influx in rat parotid acinar cells is via a loop diuretic-sensitive Na+/K+/Cl- co-transport mechanism as may occur in other secretory epithelia.     

Isoproterenol and G2 acinar cells in the developing rat parotid gland.

In the present study, premitotic period (G2)-delayed or blocked acinar cells were identified in the parotid gland of the neonatal rat. The noncycling G2 cells retain the potential to divide and respond to administration of the sympathomimetic amine, isoproterenol (IPR). The effects of IPR on suckling and weaned rats were compared. Both groups showed the presence of G2-acinar cells with a possible age-related change occurring after weaning.     

Exocyst subunits are involved in isoproterenol-induced amylase release from rat parotid acinar cells

Exocytosis of secretory granules in parotid acinar cells requires multiple events: tethering, docking, priming, and fusion with a luminal plasma membrane. The exocyst complex, which is composed of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) that are conserved in yeast and mammalian cells, is thought to participate in the exocytotic pathway. However, to date, no exocyst subunit has been identified in salivary glands. In the present study, we investigated the expression and function of exocyst subunits in rat parotid acinar cells. The expression of mRNA for all eight exocyst subunits was detected in parotid acinar cells by RT-PCR, and Sec6 and Sec8 proteins were localized on the luminal plasma membrane. Sec6 interacted with Sec8 after 5 min of stimulation with isoproterenol. In addition, antibodies to-Sec6 and Sec8 inhibited isoproterenol-induced amylase release from streptolysin O-permeabilized parotid acinar cells. These results suggest that an exocyst complex of eight subunits is required for amylase release from parotid acinar cells.     

藻酸盐诱发的免疫反应对兔腮腺腺细胞的作用

目的:探讨藻酸盐诱发的免疫反应对腮腺腺细胞的作用.方法:采用Alginate-BSA交联物免疫兔,ELISA法检测藻酸盐抗血清效价;实验分5 组:A、空白对照组, B、牛血清白蛋白组, C、藻酸盐组, D、抗藻酸盐血清组和E抗藻酸盐血清+藻酸盐组,分别在1、6、12和24 h采用MTT法检测各组腮腺腺细胞增殖情况.倒置显微镜观察不同组腮腺腺细胞生长、形态和结构变化;扫描电镜观察E组腺细胞超微结构的改变.结果:Alginate-BSA交联物免疫兔约40 d,藻酸盐抗血清的效价达到1∶ 400;二者适宜反应浓度:藻酸盐为40 μg/ml,抗藻酸盐血清的稀释度为1∶ 100;MTT检测结果A、B、C、D、E组在前3 个时间点没有差异,而在24 h时,E组与A、B、C、D组差异显著(P<0.05),说明抗藻酸盐血清和藻酸盐的免疫反应对腺细胞的增殖有显著的抑制.倒置显微镜下在12 h和24 h可观察到E组个别腺细胞表面有破裂,胞质外溢,细胞形态不完整;其它组未见异常.扫描电镜观察E组在6 h即有个别腺细胞胞膜有破裂,呈圆孔形,细胞的轮廓仍清晰完整.12 h可见破裂细胞数增多,且细胞膜上破裂孔、裂也增多变大.24 h可见细胞的形态不完整,有较大范围胞膜破裂,细胞崩解.结论:抗藻酸盐血清和藻酸盐反应对腮腺腺细胞可造成免疫损伤,导致细胞死亡.