Cells Of Rat Submandibular Gland Research Articles

Effect of PACAP-27 on adenylate cyclase in ductal and acinar cells of rat submandibular gland.

Effect of PACAP-27 on adenylate cyclase in ductal and acinar cells of rat submandibular gland.

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.

NNOS and Ca 2+ influx in rat pancreatic acinar and submandibular salivary gland cells

Regulation of agonist-activated Ca 2+ influx by the NOS pathway through generation of cGMP is being found in an increasing number of cell types. In the present work, we examined the role of the NOS pathway in agonist-evoked [Ca 2+] i oscillations and attempted to identify the NOS isoform most likely to regulate Ca 2+ influx. For this, we first show that two Ca 2+-mobilizing agonists acting on pancreatic acinar cells, bombesin (BS) and the cholecystokin in analog CCK-JMV-180 (CCKJ), evokes different type of [Ca 2+] i oscillations. The BS-evoked [Ca 2+] i oscillations rapidly became acutely dependent on the presence of extracellular Ca 2+, whereas the CCKJ-evoked oscillations continue for long periods of time in the absence of Ca 2+ influx. This differential behavior allowed us to isolate Ca 2+ influx and study its regulation while controlling for non specific effects on all other Ca 2+ transporting events involved in generating [Ca 2+] i oscillations. Inhibitors of selective steps in the NOS pathway inhibited agonist-induced cGMP production. The inhibitors were then used to show that scavenging NO with reduced hemoglobin, inhibition of guanylyl cyclase with 1 H-[1,2,4] oxadiazolo[4,3-a] quinoxaline-1-one (ODQ) and inhibition of protein kinase G with Rp-8-pCPT-cGMPS inhibited [Ca 2+] i oscillations evoked by BS but not those evoked by CCKJ. These findings were extended to duct and acinar cells of the SMG. In these cells, Ca 2+-mobilizing agonists stimulate large Ca 2+ influx, which was inhibited by all inhibitors of the NOS pathway. Western blot analysis and immunolocalization revealed that the cells did not express iNOS, eNOS was expressed only in blood vessels and capillaries whereas nNOS was expressed at high levels next to the plasma membrane of all cells. Accordingly, the nNOS inhibitor 7-nitroindazole (7-NI) inhibited BS- but not CCKJ-evoked [Ca 2+] i oscillations and Ca 2+ influx into SMG acinar and duct cells. Thus, together, our findings favor nNOS as the isoform activated by the Ca 2+ released from internal stores to generate cGMP and regulate Ca 2+ influx.

Regeneration of myoepithelial cells in rat submandibular glands after yttrium aluminium garnett laser irradiation.

The regeneration of myoepithelial cells in rat submandibular salivary gland after partial irradiation with yttrium aluminium garnett (YAG) laser was investigated. The irradiated glands were examined immunohistochemically for actin, histochemically for alkaline phosphatase (ALP), and by transmission electron microscopy (TEM). In control glands, myoepithelial cells were positive for actin and ALP. Electron microscopically, the positive reaction for actin was associated with the myofilaments of myoepithelial cells, and the plasma membrane of myoepithelial cells was positive for ALP. One day after YAG laser irradiation, the irradiated region was necrotic. By 5 days, duct-like structures and epithelial clusters were observed at the interface between the necrotic zone and the remaining undamaged glands; immature acini appeared after 7 days. No reaction in duct-like structures or epithelial clusters to actin or ALP was recognizable by 5 days. However, at 7 days, actin and ALP-positive spindle cells appeared at the periphery of the duct-like structures and immature acini. After 10 days, both actin-positive and ALP-positive cells increased in number. These observations indicate that during regeneration, actin-positive and ALP-positive cells regenerate myoepithelial cells, and it is suggested that this differentiation to myoepithelial cells is closely related to that of luminal to acinar cells. In addition, TEM observations indicate that regenerated myoepithelial cells originated from the basal cells of duct-like structures.

Studies on the coated vesicle-associated filament appearing during granule formation by acinar cells of rat submandibular gland.

Studies on the coated vesicle-associated filament appearing during granule formation by acinar cells of rat submandibular gland.

X-ray microanalysis applied to the study of salivary glands.

X-ray microanalysis can be used to study the distribution of elements in salivary glands at the cellular and subcellular level in a variety of physiological and pathophysiological conditions. The acinar cells of the adult rat and mouse submandibular gland contain high concentrations of calcium due to the presence of calcium-rich secretory granules. The calcium concentration of the acinar cells can be increased under a variety of pathophysiological conditions, e.g., in animal models for cystic fibrosis, due to an increase in the number of secretory granules and changes in their calcium-binding properties. Cholinergic stimulation results in a decrease in the concentrations of Cl and K in all cell compartments in adult animals, but not in newborn rats. Also the secretory granules in the glands in adult animals lose Cl and K, indicating that ion (and water) transport mechanisms may be present in the membrane of the granules. During chemical fixation, the secretory granules in the adult glands swell and their morphology is different from that in freeze-substituted glands. The characteristics of the secretory granules in the acinar cells of newborn rats are different from those of adult animals.

An antagonist-insensitive P2X receptor expressed in epithelia and brain.

A cDNA was cloned which encodes a new ATP-gated ion channel (P2X4 receptor). ATP induces a cationic current in HEK293 cells transfected with the P2X4 receptor. However, the current is almost completely insensitive to antagonists effective at other P2X receptors. Sensitivity to two of these antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and pyridoxal 5-phosphate) is restored by replacement of Glu249 by lysine, which occurs at the equivalent position in P2X1 and P2X2 receptors. P2X4 RNA is found by in situ hybridization in the brain, peripheral ganglia and epithelia including serosal cells of salivary glands. Recordings from rat submandibular gland cells showed ATP-induced currents that are also insensitive to antagonists. These results define a further member of P2X receptor family, and they identify an amino acid residue involved in antagonist binding. They also introduce a new phenotype for ATP responses at P2X receptors--insensitivity to currently known antagonists.

Distribution and translocation of isoforms of protein kinase C in rat submandibular acinar cells

The distribution of six isoforms of protein kinase C (PKC) in seromucous acinar cells of rat submandibular gland was examined and their translocation from the cytosolic- to the membrane fraction after different stimuli investigated. Western blotting, immunostaining with isoform-specific antibodies and scanning densitometry showed that PKC-α and ϵ were distributed fairly evenly between the cytosol and membranes in resting cells, while isoforms- β, δ and ζ were all predominantly localized (over 80%) in membranes. PKC-γ was not detected. PKC-α was mobilized to the membrane fraction by the phorbol ester, TPA, but not by the phosphoinositide-coupled agonists carbachol, methoxamine and substance P (SP). PKC-ϵ was translocated by TPA and carbachol but not by SP or methoxamine. Biochemical assay of total PKC confirmed that cytosolic enzyme activity was significantly reduced by TPA and carbachol to 29% and 75% respectively of control levels. These results suggest that muscarinic regulation of the mucosecretory response in the rat submandibular gland may be mediated by the PKC-ϵ isoform.

Monoclonal antibody against the glutaraldehyde-conjugated polyamine, spermine

We developed a mouse monoclonal antibody (ASPM-29, mAb) against spermine (Spm) conjugated to human serum albumin (HSA) using glutaraldehyde-sodium borohydride, for applications in immunocytochemistry (ICC). The antibody specificity was evaluated by an enzyme-linked immunosorbent assay (ELISA) binding test, simulating the ICC of tissue sections. ASPM-29 showed an almost equal immunoreactivity to Spm and spermidine (Spd) but no reactivity to any of the other polyamine (PA)-related compounds tested. By use of this antibody, indirect immunoperoxidase staining was observed in different tissues fixed with glutaraldehyde in combination with borohydride reduction. In contrast, immunoreactivity was quite low in tissues fixed only with glutaraldehyde. Absorption controls indicated that the immunostaining could be completely inhibited by 50 micrograms/ml of Spm or Spd and partially inhibited by N-acetylspermine (Ac-Spm), N1-acetylspermidine (N1-Ac-Spd), or N8-acetylspermidine (N8-Ac-Spd), but was hardly inhibited at all by other PA-related compounds or amino acids. The reactivity of the antibody with Spm conjugated on wells in an ELISA plate was inhibited by micromolar concentrations of Spm, Spd, Ac-Spm, N1-Ac-Spd, or N8-Ac-Spd, in decreasing order, but not by other small molecules. Dense ICC staining was observed in the paranuclear and basal cytoplasm of acinar cells of rat pancreas, submandibular gland and paratid gland, these results being in complete agreement with our recent ICC methods using other mAbs produced against N-(gamma-male-imidobutyryloxy) succinimide-conjugated Spm.

Arachidonic Acid Stimulates Intracellular Calcium Mobilization and Regulates Protein Synthesis, ATP Levels, and Mucin Secretion in Submandibular Gland Cells

Earlier observations that arachidonic acid inhibited the synthesis of membrane inositol phospholipids in rat submandibular acinar cells prompted the present study on whether the fatty acid may also regulate other key physiological processes in the model. Arachidonate, at concentrations above 10 mumol/L, inhibited up to 97% protein synthesis in acinar cells. The acid also lowered cellular ATP levels to 25% of control values by a ouabain-insensitive mechanism. In endoplasmic reticulum-calcium studies in permeabilized cells, arachidonic acid stimulated the mobilization of up to 73% loaded ER-45Ca2+ to the cytosol, a much greater response than those caused by other calcium translocators, thapsigargin or inositol 1,4,5-trisphosphate. Additionally, arachidonate provoked the release of over 80% of total cell 45Ca2+ to the extracellular space in intact cells and stimulated mucin secretion in the submandibular model. The inhibitory effect of arachidonic acid on protein synthesis was duplicated by carbachol, thapsigargin, and BAPTA/AM, three agents that cause net efflux of ER-Ca2+ by different mechanisms. Furthermore, comparable with the arachidonate effect on ATP, carbachol and thapsigargin also significantly reduced cellular levels of the nucleotide. It is concluded that arachidonic acid acts as a regulator of central synthetic/secretory processes in mucous acinar cells of rat submandibular gland and suggested that at least some of its effects may be secondary to its calcium-mobilizing action.

Regulation of phosphatidylinositol kinases by arachidonic acid in rat submandibular gland cells

Phosphoinositide kinases were characterized in membrane extracts of rat submandibular gland cells. Both phosphatidylinositol (PI) 4-kinase and phosphatidylinositol-4-phosphate (PI(4)P) 5-kinase phosphorylated endogenous substrates in reactions that were linear for up to 5 min, were activated by Mg2+ and showed maximal activity around neutral pH. PI 4-kinase was stimulated by Triton X-100 at an optimal concentration of 0.22%, but the detergent had an inhibitory effect on PI(4)P 5-kinase. Arachidonic acid (AA), at concentrations greater than 100 microM, inhibited the activity of both enzymes in a dose-dependent manner. The inhibitory effect was replicated by other unsaturated fatty acids, but not by a saturated fatty acid of the sn-20 series. The nature of AA inhibition of the kinases was examined in enzyme kinetic studies with exogenous phosphoinositide and adenosine 5'-triphosphate (ATP) substrates. Lineweaver-Burk plots of PI 4-kinase activity showed that AA had no effect on the apparent Km for either PI or ATP, but that the fatty acid significantly reduced Vmax (PI) from 331 to 177 pmol.mg-1.min-1 and Vmax (ATP) from 173 to 59 pmol.mg-1.min-1. This inhibitory action was consistent for PI(4)P 5-kinase kinetics, where again, AA did not alter apparent Km values, but lowered Vmax for both PI(4)P and ATP by around 50%. Since the combination of a reduced Vmax and an unchanged Km value indicates noncompetitive enzyme inhibition, it is proposed that AA regulates phosphoinositide cycle activity in submandibular gland cells by acting as a noncompetitive inhibitor of PI 4-kinase and PI(4)P 5-kinase.

Delayed inhibition of gap-junctional intercellular communication in the acinar cells of rat submandibular glands induced by parasympathectomy and cholinergic agonists

In this study, the effects of parasympathectomy and cholinergic agonists on gap-junctional intercellular communication and salivary secretion were investigated to clarify the involvement of salivary secretion in delayed uncoupling between acinar cells of rat submandibular glands. Gap-junctional intercellular communication was monitored as dye-coupling in the acinar cells of isolated acini by the transfer of Lucifer Yellow CH. Parasympathectomy induced dye-uncoupling in the acinar cells isolated from denervated salivary glands 12 hr after parasympathectomy-induced salivary secretion. Intraperitoneal application of carbachol (CCh), acetylcholine, pilocarpine, but not isoproterenol, stimulated salivary secretion, and then induced dye-uncoupling in the acinar cells 12 hr later. Atropine suppressed both the salivary secretion and delayed dye-uncoupling induced by parasympathectomy and CCh, when atropine was applied intraperitoneally before the induction of salivary secretion. However, atropine did not suppress the delayed dye-uncoupling by intraperitoneal application of CCh, when atropine was injected after the cessation of CCh-induced secretion. These results suggest that delayed inhibition of gap-junctional intercellular communication by parasympathectomy and cholinergic agonists in rat submandibular glands might be related to the change of secretory function after salivary secretion.

Stimulated Ca2+ entry activates Cl- currents after releasing Ca2+ from the intracellular store in submandibular gland cells of the rat.

In order to examine whether Ca2+ entry is directly involved in controlling exocrine secretion, the Ca(2+)-activated Cl- currents were recorded in single and clusters of rat submandibular gland cells using the whole-cell patch-clamp method. Extracellularly applied acetylcholine (ACh, 10 nM) as well as intracellularly applied GTP gamma S and InsP3 caused repetitive transients of the Cl- currents activated by intracellular Ca2+. These responses occurred also in the absence of external Ca2+, but disappeared after several minutes. Readmission of Ca2+ to the extracellular solution restored the repetitive current transients, while introduction of Sr2+ failed to restore the current signals in spite of the presence of Sr2+ entry detected by microfluorimetry. On the other hand, direct application of Sr2+ to the cell inside caused activation of the Cl- currents although less effectively than Ca2+. When Ca2+ was introduced to the extracellular solution during an interruption of ACh stimulation after the ACh-induced depletion of intracellular Ca2+ store, the Cl- current was not elicited. However, a subsequent challenge with ACh at the same concentration in the absence of extracellular Ca2+ caused repetitive transient Cl- currents. The results suggest that in this cell type the stimulated Ca2+ entry does not by itself activate the Cl- currents but activates them indirectly by triggering Ca2+ release from the intracellular Ca2+ store which may take up Ca2+ soon after the Ca2+ entry.

The fate of glycogen in granular tubule cells of rat submandibular glands during secretory events

Glycogen, studied electron microscopically in granular tubule cells by means of the periodic acid-thiocarbohydrazide-silver proteinase technique, was found to be scattered abundantly throughout the cytoplasm. Parasympathetic nerve stimulation caused no detectable change in the glycogen. Degranulation of the granular tubule cells after either sympathetic nerve stimulation or cyclocytidine injection caused loss of the glycogen from the cells. Study of tubule cells undergoing secretion in the early stages after cyclocytidine injection indicated that glycogenolysis was occurring. Glycogen had reaccumulated in the cells within 24 h, before extensive reformation of the secretory granules had occurred, and remained abundant throughout the subsequent granule formation. It is concluded that glycogen provides an important source of energy during secretory degranulation of the granular tubule cells.

Localization of mRNAs of two androgen-dependent proteins, SMR1 and SMR2, by in situ hybridization reveals sexual differences in acinar cells of rat submandibular gland.

Androgen-dependent sexual differences in the granular convoluted tubules of mouse and rat submandibular glands (SMG) have been extensively reported. We studied two major androgen-dependent mRNAs of the rat SMG encoding proteins named SMR1 and SMR2. To determine which cell type in the SMG is responsible for synthesis of these mRNAs, we performed in situ hybridization with digoxigenin-labeled RNA probes coupled with alkaline phosphatase detection. We show that SMR1 and SMR2 mRNAs are synthesized in the acinar cells of the SMG. A clear difference in SMR1 and SMR2 mRNA levels in male and female is demonstrated. During the course of this study we also confirmed the acinar localization of mRNAs encoding the glutamine/glutamic acid-rich proteins (GRP) of rat SMG. Our data are the first clear evidence of androgen-dependent sexual differences in acinar cells of rat submandibular gland.

Chronoimmunohistochemical localization of epidermal growth factor in the submandibular gland of male rats

Chronobiological oscillation was established for epidermal growth factor (r-EGF) in granular convoluted tubule (GCT) cells of the male rat submandibular gland (SMG) using immunohistochemistry. The high amplitude oscillation with a peak at 9 p.m. and low amplitude oscillation during the day time was noted. The distribution pattern of the r-EGF in the GCT segment was time-dependent, as it was seen at 3 p.m. when many cells with r-EGF staining were detectable. The higher reactivity during the activity phase of the rats may correlate with the intracellular biosynthesis of r-EGF required for cell proliferation and maintenance of ductal integrity. The chronobiological oscillation pattern of r-EGF seems to be important for the maintenance of ductal integrity and wound healing.

The B1-immunoreactive proteins of the perinatal submandibular gland: similarity to the major parotid gland protein, RPSP.

The B1-immunoreactive proteins of type III cells of the perinatal rat submandibular gland are immunologically cross-reactive with proteins of both the sublingual and parotid glands; in particular, protein SMG-A appears similar to a major parotid protein. We isolated SMG-A and the parotid protein (known as M1 or leucine-rich protein), prepared polyclonal antibodies to them, and compared their biochemical properties and immunological reactivities. They were identical in their molecular weight on SDS-PAGE (23.5 kDa), tenacious binding to Affi-gel Blue, isoelectric point (pH 4.53), and proteolysis to a 14 kDa peptide: Antibodies to SMG-A showed reactivity with protein SMG-C, a product of the neonatal type I cells, as well as with proteins SMG-B1 and SMG-B2, contrasted with the absence of reactivity of anti-M1 IgG with these proteins. Anti-M1 reacted with the "parotid secretory protein" (PSP) of the mouse, and M1 appears to be the homologue, in the rat, of mouse PSP.

Distribution of anionic sites during increasing tight junctional permeability in the rat submandibular gland.

We sought to determine the effect of substance P salivary stimulation on both electrical charges and cellular permeability in the tight junctions of rat submandibular gland cells. Microperoxidase (1,900 daltons) was used as a tracer. It was administered by close-arterial infusion via the glandular arteries, and secretory routes of acinar cells in the gland were determined cytochemically. In the resting gland, microperoxidase reaction product filled the lateral intercellular spaces up to the tight junctions, but did not penetrate them. In the substance P-stimulated gland, microperoxidase reaction product was present within tight junctions and the lumen. Both distribution and mobility of anionic sites on the surface of the submandibular gland cells were studied utilizing multivalent ligand, ruthenium red and cationized ferritin as probes. In the resting gland, ruthenium red deposits were located uniformly in all areas of the basal membrane and intercellular spaces except for the tight junctional region of acinar and ductal cells. In substance P-stimulated gland, ruthenium red deposits were present in the tight junctional region and, to a lesser extent, in the intercellular spaces. Electrical charges of the tight junctions area of the lateral plasma membrane were studied using intraductal injection of cationized ferritin. In the resting gland, cationized ferritin probe was present in the intercellular spaces and was bound weakly in the tight junctional region. In the substance P-stimulated gland, cationized ferritin was firmly adherent to the tight junctional region.

Accumulation and localization of two adult acinar cell secretory proteins during development of the rat submandibular gland

The seromucous acinar cells of the adult rat submandibular gland secrete a characteristic mucin glycoprotein and a family of unusual glutamine/glutamic acid-rich proteins (GRP). Monoclonal antibodies to the mucin and GRP localized in a very few Type III cells in glands of newborn and 1 day-old rats, using light and electron microscopic immunocytochemistry. Both mucin and GRP reactivities were present in the polymorphic Type IIIP granules during the 1st postnatal week. By 9 days after birth, the granules contained both mucin and GRP and were mucous-like in appearance. At earlier stages, however, cells containing only GRP or mucin could be found, indicating that the initiation of GRP and mucin biosynthesis may not be coordinately regulated. No reactivity was seen in the neonatal Type I cells or in duct cells at any age. Northern and Western blot analysis showed GRP mRNA and protein levels to be barely detectable at birth, with marked increases during the first 2 postnatal weeks. In contrast, Western blots of B1-immunoreactive proteins (B1-IP) showed levels highest in the 1st week and markedly decreased in the adult. Immunocytochemical colocalization, using gold particles of different sizes, showed that the B1-IP, mucin, and GRP colocalized in the granules. These results strengthen the hypothesis that the adult acinar cells develop from the neonatal Type III cells. No evidence was obtained for the involvement of Type I cells in the pathway of acinar cell development.