Rat Salivary Gland Cells Research Articles

Intravital Microscopy Reveals Differences in the Kinetics of Endocytic Pathways between Cell Cultures and Live Animals

Intravital microscopy has enabled imaging of the dynamics of subcellular structures in live animals, thus opening the door to investigating membrane trafficking under physiological conditions. Here, we sought to determine whether the architecture and the environment of a fully developed tissue influences the dynamics of endocytic processes. To this aim, we imaged endocytosis in the stromal cells of rat salivary glands both in situ and after they were isolated and cultured on a solid surface. We found that the internalization of transferrin and dextran, two molecules that traffic via distinct mechanisms, is substantially altered in cultured cells, supporting the idea that the three dimensional organization of the tissue and the cues generated by the surrounding environment strongly affect membrane trafficking events.

In vitro photosensitization initiated by camphorquinone and phenyl propanedione in dental polymeric materials

Documentation is scarce on the photobiological effects of photoinitiators present in dental light curable materials. The aim of this study was to determine cellular effects of the photoinitiators camphorquinone (CQ) and phenyl propanedione (PPD) and to investigate whether these substances produced reactive oxygen species after low and high doses of optical radiation (between 0 and 17 J/cm 2). Rat salivary gland cells in vitro were exposed to visible blue light and/or UVA. Hematoporphyrin (HP), a photosensitizer used in medicine, and the UVA-filter 2-methoxy-4-hydroxy-benzophenone (B-3) were used as reference substances. It was found that PPD produced hydrogen peroxide, but not singlet oxygen, upon light irradiation. CQ produced neither hydrogen peroxide nor singlet oxygen. Cell death by necrosis and apoptosis was induced by irradiation in the presence of CQ, PPD and HP. Doses higher than 6 J/cm 2 UVA and blue visible light from a source similar to clinically applied sources, induced apoptosis even in the absence of photosensitizers added. A reciprocity relationship was found between radiant exposure (at constant irradiance) and concentration of photoinitiators. In conclusion, the oral cells under investigation were light sensitive, and the sensitivity increased in presence of photoinitiators. PPD acted by mechanisms that included reactive oxygen species and CQ probably by formation of free radicals.

Dispersed donor salivary gland cells are widely distributed in the recipient gland when infused up the ductal tree

The salivary glands often are severely and permanently damaged by therapeutic irradiation for cancer of the head and neck. The markedly reduced quantity and quality of saliva results in greatly increased susceptibility to dental caries and infection of the oral mucosa and alveolar bone. Recently, subcapsular injection of cultured mouse salivary gland cells has achieved a significant degree of regeneration in a previously irradiated mouse salivary gland; however, the recovery was limited to one lobule. We describe here a method for delivering donor rat salivary gland cells via the main duct that distributes several thousand cells throughout the recipient rat's salivary gland. The donated cells exhibited the cytodifferentiation of the structures in which they lodged, i.e., acini, granular convoluted tubules, and the several types of ducts. This method may facilitate the simultaneous functional recovery of almost all of the lobules of irradiated rat salivary glands.

Characterization of CLCA protein expressed in ductal cells of rat salivary glands

A molecular entity for Ca 2+-dependent Cl − transport has not been well characterized in salivary cells. Here, we identify a rat CLCA homologue (rCLCA1) using a polymerase chain reaction (PCR)-based strategy. The full length of the isoform was 3.3 kb, and the predicted open reading frame encoded a 903-amino acid protein. Immunoblotting using a specific anti-rCLCA antibody recognizing near the amino-terminus showed the expression of N-glycosylated 120- and 86-kDa proteins in the membrane fraction of rCLCA1-transfected HEK293 cells. Reverse transcription-PCR results showed mRNA expressions in rat submandibular gland (SMG), ileum, and lung. Intense immunostaining was detected in the striated ducts, but not in the acinar cells, of SMG. Immunoblot for the membrane fraction of SMG revealed the existence of 137- and 90-kDa protein species. N-glycosidase F reduced the size of these bands toward those of the deglycosylated forms in the transfected HEK293 cells. A marked ionomycin-induced Cl − conductance was observed in the transfected cells. The current was Ca 2+-dependent and sensitive to niflumic acid and DIDS. rCLCA1 proteins are probably responsible for modulation of Ca 2+-dependent Cl − transport in salivary ductal cells, where the 137- and 90-kDa proteins may be modified posttranslationally in a manner similar to those in the heterologous expression system.

Amperometric determination of choline released from rat submandibular gland acinar cells using a choline oxidase biosensor

A choline (CHO) biosensor based on the determination of H 2O 2 generated at the electrode surface by the enzyme choline oxidase (CHOx) was developed. The biosensor consisted of CHOx retained onto a horseradish peroxidase (HRP) immobilized solid carbon paste electrode (sCPE). The HRPsCPE contained the molecule phenothiazine as redox mediator and CHOx was physically retained on the electrode surface using a dialysis membrane. Several parameters have been studied such as, mediator amount, influence of applied potential, etc. The CHO measurements were performed in 0.1 M phosphate buffer, pH 7.4. Amperometric detection of CHO was realized at an applied potential of 0.0 mV vs Ag/AgCl. The response is linear over the concentration range 5.0×10 −7–7.0×10 −5 M, with a detection limit of 1.0×10 −7 M. This biosensor was used to detect choline released from phosphatidylcholine (PC) by phospholipase D (PLD) in isolated rat salivary gland cells stimulated by a purinergic agonist (ATP).

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.

Upregulation of P2Y2 nucleotide receptors in rat salivary gland cells during short-term culture.

In contrast to the widespread expression of G protein-coupled P2Y2 receptors for extracellular nucleotides in permanent cell lines of salivary gland origin, there is less evidence for robust P2Y2 receptor activity in normal rat salivary gland cells assayed immediately after isolation. We examined the effect of short-term culture (3 h to 6 days) of normal rat submandibular gland (SMG) cells on P2Y2 receptor activity and mRNA expression. Results indicate that increases in the intracellular free Ca2+ concentration in SMG cells in response to the P2Y2 receptor agonist UTP (100 microM) were detectable after 3 h in culture and that after 3 days in culture the magnitude of the response to UTP was similar to that obtained with maximal muscarinic cholinoceptor activation. The Ca2+ mobilization response exhibited the pharmacological profile (UTP = ATP > 2-methylthioadenosine 5'-triphosphate) typical of the P2Y2 receptor subtype and was accompanied by enhanced production of inositol phosphates, reflecting the activation of phospholipase C ubiquitously associated with P2Y2 receptors. The time-dependent increase in P2Y2 receptor activity was accompanied by an increase in the steady-state level of P2Y2 receptor mRNA, as assessed by reverse transcription-polymerase chain reaction. Other studies revealed that the increased P2Y2 receptor activity was independent of cell proliferation, was similar in serum-containing and defined culture media, and was blocked by inhibitors of transcription and translation. Upregulation of the P2Y2 receptor was observed in both acinar cell- and ductal cell-enriched cultures of the SMG and in cells isolated from rat parotid and sublingual glands but not in cells isolated from the pancreas. These in vitro results were complemented by in vivo studies in which P2Y2 receptor activity and mRNA levels were increased in SMG after ligation of the main excretory duct but were not increased in the contralateral, nonligated gland. These findings suggest that changes in the expression and activity of the P2Y2 receptor in salivary gland cells may be related to pathological challenges to the gland in vivo.

ラット唾液腺核蛋白質のりん酸化の加齢に伴う動態

Development, growth, maturation and aging processes of secretory cells of rat salivary glands progress mainly after birth. Nuclear non-histone proteins, phosphorylated actively and reversively, have an important role as regulatory molecules of gene activity and have a possibility to bring about specific changes in these cellular processes. We examined in the present study the age-dependent changes in the phosphorylation of non-histone proteins of rat salivary glands. Nuclei purified from submandibular and parotid glands of 8-week-old rats rapidly incorporated 32P from gamma-32P-ATP into the nuclear phosphoproteins and reached equilibrium within 9 min. A preponderant amount of the 32P was present in non-histone proteins. The levels of phosphorylation of non-histone proteins in salivary gland nuclei increased rapidly after birth, reaching a maximum in both gland nuclei of 4-week-old rats and then decreasing to the levels observed in submandibular and parotid gland nuclei from 20 and 16-week-old rats, respectively. These levels were still maintained in nuclei from aged rats. Moreover, age-dependent changes in the protein kinase activity of submandibular and parotid gland nuclei were linked up with the changes in the phosphorylation of non-histone proteins. However, changes were not observed in the phosphorylation of histone proteins after birth. These results suggest that protein kinase activity in salivary gland nuclei may have an important role on age-dependent changes in cell function, mediated through the control of the phosphorylation of non-histone proteins.

Calcium ion uptake induced by cholinergic and ?-adrenergic stimulation in isolated cells of rat salivary glands

Adrenaline (10(-5) M) and carbamylcholine (10(-4) M) stimulate 45Ca2+ uptake into isolated cells of rat submandibular galnd and parotid glands. In the presence of the alpha-adrenoreceptor blocking agent phentolamine, adrenaline stimulation of 45Ca2+ uptake is abolished. The beta-adrenergic stimulant isoproterenol has no effect on 45Ca2+ uptake. Carbamylcholine induced 45Ca2+ uptake is inhibited by atropine. The Ca2+ ionophore A23187 stimulates 45Ca2+ uptake, whereas dibutyryl cyclic adenosine 3',5'-monophosphate and dibutyryl cyclic guanosine 3',5'-monophosphate have no effect on 45Ca2+ uptake. A graphical analysis of the 45Ca2+ uptake curves reveals at least two phases: a fast phase and a slow phase, both of which are stimulated by adrenaline and carbamylcholine. The 45Ca-exchangeable pool size is increased by adrenaline and carbamylcholine in both the fast and the slow phases. These results suggest that alpha-adrenergic and cholinergic agonists act by increasing the rate of Ca2+ transfer into the cells of the parotid and submandibular salivary glands most probably through an increase of the cell membrane permeability for Ca2+.

Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding. I. Rate of (glyco) protein secretion from cells of mixed salivary glands.

In untreated, fasting animals the cells of the serous demilunes of the sublingual gland incorporate [3H]-leucine at a higher rate than any other of the 5 main cell types of the 3 major salivary glands. The acinar cells of the submandibular and the mucous cells of the sublingual gland show intermediate values, while the cells of the granular ducts of the submandibular and the acini of the parotid gland have a low rate of incorporation. In fasting animals extrusion of newly synthesized protein starts early in the cells of the serous demilunes. It starts between 4 and 7 hrs after [3H]-leucine injection in the acinar cells of the submandibular gland, while the other cell types did not lose substantial amounts of labelled (glyco)protein within 7 hrs. The secretion of protein is stimulated by the cholinergic drug pilocarpine in all but one of the 5 types of salivary gland cells studied. The acinar cells of the submandibular gland react strongly, the granular duct cells less strongly. Still less are the reactions of the acinar cells of the parotid and of the nucous cells of the sublingual gland. The cells of the serous demilunes of the latter appear to be insensible to pilocarpine. The effect of food uptake on secretion does not differ from pilocarpine stimulation, with one exception: the acinar cells of the parotid gland react more strongly on food uptake than on cholenergic stimulation.

Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding. II. Protein synthesis in vivo in cells of the submandibular and parotid gland after secretion.

The effect of pilocarpine and food uptake on the rate of incorporation of [3H]-leucine in vivo was measured by means of quantitative radioautography in three exocrine cells of the rat: the acinar and the granular duct cells of the submandibular and the acinar cells of the parotid gland. The three cell types react differently. The submandibular acinar cells showed a decrease in incorporation rate after pilocarpine administration but not after feeding. The incorporation rate of the granular duct cells of the submandibular gland remains constant after both stimulations. The acinar cells of the parotid gland show an increase in incorporation rate of [3H]-leucine in response to both. The contrast between the submandibular and the parotid gland could also be demonstrated radiobiochemically, the results reflecting the incorporation rates of the acinar cells of both glands, giving no information on the contribution of other cell types. The decrease in incorporation rate of the submandibular gland acinar cells is accompained by a shift of polyribosomes towards monomers.

Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding. III. Protein synthesis in vitro in the submandibular and parotid gland after stimulation of secretion in vivo.

After stimulation of the protein secretion by pilocarpine or feeding the rate of incorporation of [3H]-leucine increases in the acinar cells of the parotid gland of the rat while the secretory cells of the submandibular gland show a moderate decrease (Kuijper et al., 1975b). Since the rate of labelled amino acid incorporation depends on the specific radioactivity of the amino acid used, which is not easy to determine in vivo, experiments in vitro were performed to get an idea of the influence of this factor on the measured changes in [3H]-leucine incorporation. In vitro both cell types showed a more pronounced but essentially identical reaction as in vivo. Since in these experiments the specific radioactivity of the extracellular leucine is the same whether fragments of stimulated or unstimulated glands incorporate the radioactive amino acid, the increase of incorporation in the parotid and the decrease in the submandibular cells cannot be ascribed to differences in specific radioactivity of leucine, unless the intracellular leucine pool should show great differences between secreting and non-secreting cells. However, in vitro the submandibular gland cells under both conditions appear to use the extracellular leucine for their protein synthesis (or a small compartmentalized pool in rapid exchange with the extracellular pool). In the parotid cells the whole intracellular pool showed such a rapid exchange with the extracellular one that for practical reasons one may say that these cells, too, rely on the extracellular specific radioactivity of leucine in their protein synthesis. We conclude that the rat parotid gland cells show a rapid and substantial increase of protein synthesis after stimulation of their enzyme secretion, while the submandibular gland cells do not.

Membrane potentials of salivary gland cells of rat.

Transmembrane electrical potentials were recorded from cells of rat salivary glands in vivo, using microelectrodes. The mean (±se) from 61 impalements of unstimulated submaxillary cells was 28 ± 1.4 mv. In submaxillary gland, acinar cells and cells of granular tubules account for approximately 70 and 30%, respectively, of all secretory elements. Dividing the range of submaxillary potentials at 35 mv gave two groups with new means of 23 ± 1.0 and 41 ± 0.6 mv, in which were contained 70 and 30%, respectively, of all values. Potentials in submaxillary gland from which granular tubules had been caused virtually to disappear (by isoproterenol) were always below 35 mv (mean ± se = 25 ± 0.6 mv). Similarly, potentials from normal parotid gland (in which granular tubules do not appear) never exceeded 34 mv (mean ± se = 20 ± 2.4). In submaxillary, stimulation, at least over relatively long durtion, produced no change in average potential. It was concluded that membrane potentials from rat salivary glands are generally below the potassium equilibrium potential, with acinar cells showing lower values than cells of granular tubules.

HISTOCHEMICAL LOCALIZATION OF ALKALINE PHOSPHATASE AND ADENOSINE TRIPHOSPHATASE IN THE MYOEPITHELIAL CELLS OF RAT SALIVARY GLANDS.

THE histochemical demonstration of alkaline phosphatase in the myoepithelial cells of salivary glands, sweat glands and mammary glands has been reported by a number of workers1–7.