Rat Submandibular Gland Acinar Cells Research Articles

Ca2+-CaMKKβ pathway is required for adiponectin-induced secretion in rat submandibular gland.

Adiponectin functions as a promoter of saliva secretion in rat submandibular gland via activation of adenosine monophosphate-activated protein kinase (AMPK) and increased paracellular permeability. Ca2+ mobilization is the primary signal for fluid secretion in salivary acinar cells. However, whether intracellular Ca2+ mobilization is involved in adiponectin-induced salivary secretion is unknown. Here, we found that full-length adiponectin (fAd) increased intracellular Ca2+ and saliva secretion in submandibular glands. Pre-perfusion with ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) combined with thapsigargin (TG), an endoplasmic reticulum Ca2+-ATPase inhibitor, abolished fAd-induced salivary secretion, AMPK phosphorylation, and enlarged tight junction (TJ) width. Furthermore, in cultured SMG-C6 cells, co-pretreatment with EGTA and TG suppressed fAd-decreased transepithelial electrical resistance and increased 4-kDa FITC-dextran flux responses. Moreover, fAd increased phosphorylation of calcium/calmodulin-dependent protein kinase (CaMKKβ), a major kinase that is activated by elevated levels of intracellular Ca2+, but not liver kinase B1 phosphorylation. Pre-perfusion of the isolated gland with STO-609, an inhibitor of CaMKKβ, abolished fAd-induced salivary secretion, AMPK activation, and enlarged TJ width. CaMKKβ shRNA suppressed, whereas CaMKKβ re-expression rescued fAd-increased paracellular permeability. Taken together, these results indicate that adiponectin induced Ca2+ modulation in rat submandibular gland acinar cells. Ca2+-CaMKKβ pathway is required for adiponectin-induced secretion through mediating AMPK activation and increase in paracellular permeability in rat submandibular glands.

Microarray analysis of gene expression of bone marrow stem cells cocultured with salivary acinar cells

Our previous work has demonstrated that rat bone marrow stem cells (BMSCs) can transdifferentiate into α-amylase-producing cells after coculture with rat submandibular gland acinar cells. These transdifferentiated cells may be used for regeneration of damaged salivary gland. The purpose of this study was to investigate the global gene expression of rat BMSCs cocultured with rat submandibular gland acinar cells and the factors inducing this transdifferentiation. Rat BMSCs were indirectly cocultured with rat submandibular gland acinar cells by using the double chamber system for 5 and 10 days. The global gene expression of BMSCs during transdifferentiation into acinar cells was investigated by microarray analysis. A total of 45,018 probes were used and 41,012 genes were detected. After coculture for 5 days, 1409 genes were upregulated more than twofold and 1417 genes were downregulated more than twofold (p<0.005). Moreover, after coculture for 10 days, 1356 genes were upregulated more than twofold and 1231 genes were downregulated more than twofold (p<0.005). Bone morphogenetic protein (BMP)-6 was one of the top-ranked upregulated genes. The hub genes were interleukin-6 and CCAAT/enhancer-binding protein β (CEBPB) in the early and late response gene groups, respectively. This is believed to be the first study on the global gene expression of rat BMSCs cocultured with rat acinar cells. Many genes related to the function of salivary acinar cells such as those responsible for the production of α-amylase protein were upregulated and many genes related to the differentiation of BMSCs into adipocytes and osteoblasts were downregulated. In addition, BMP-6 gene was found to be highly upregulated. We proposed that three target genes, BMP-6, interleukin-6 and CEBPB, play important roles in the transdifferentiation of BMSCs into acinar cells, and are worthy of further investigation.

In vivo secretion of the mouse immunoglobulin G Fc fragment from rat submandibular glands

Salivary glands have been proposed as target organs for gene therapy. They secrete endogenous, as well as transgenic proteins, in a polarized manner. Transgene-encoded regulated pathway proteins primarily follow the regulated pathway in rat salivary glands and are secreted into saliva in an exocrine manner. Conversely, constitutive pathway proteins generally are secreted more basolaterally and thus follow the endocrine route. In the present study, we studied in vivo the sorting of the mouse immunoglobulin G2b Fc fragment, which is physiologically secreted via the constitutive pathway. Adenoviral vectors encoding the Fc fragment and human growth hormone were delivered into rat and mouse submandibular glands in vivo to compare their serum-to-saliva distribution. We also compared the intracellular localization of the Fc fragment and growth hormone by confocal microscopy. We found that the Fc fragment was secreted almost entirely into the bloodstream from rat and mouse submandibular glands via a constitutive or constitutive-like pathway. This sorting behaviour is clearly different from that of transgenic human growth hormone, which is secreted in a regulated pathway, both in neuroendocrine cells and as a transgenic protein from salivary gland cells. We also found that simultaneously expressed human growth hormone and the mouse Fc fragment do not appear to influence each other's sorting behaviour. The Fc fragment showed a primarily basal localization, whereas growth hormone showed an apical localization, in rat submandibular gland acinar cells. The results obtained in the present study indicate that the mouse Fc fragment is a useful model protein for examining the basolateral versus apical secretory pathways employed by transgenic secretory proteins in salivary glands.

Further evidence for AQP8 expression in the myoepithelium of rat submandibular and parotid glands

Previously (Wellner et al., Pflugers Arch 441:49-56, 2000) we suggested that the localization of the aquaporins (AQPs) AQP5 and AQP8 in the apical and basolateral membranes of rat submandibular gland (SMG) acinar cells, respectively, provides for transcellular water flow during saliva formation. While the localization of AQP5 in this gland has been verified in several laboratories, there have been differing reports regarding AQP8 localization. Other investigators subsequently reported that AQP8 is not expressed in the acinar or ductal cells of the major salivary glands of the rat, but in the myoepithelium of each gland. Thus, we have carried out additional studies: (1) to reassess the localization of AQP8 in the rat SMG and (2) to assess the localization of AQP8 in the rat parotid gland (PG). Initially, we compared the localizations of AQP8 with recognized basolateral markers in acinar cells [the Na+,K+-ATPase and the Na+-K+-2Cl- cotransporter (NKCC1)]. Our results indicated that Na+,K+-ATPase localized in both the basal and lateral membranes of rat SMG acinar cells, whereas AQP8 was detected only in the basal regions of the acini. In the rat PG, AQP8 was invested near intercalated ducts and adjacent acini, whereas NKCC1 localized in the basolateral membranes of acinar cells. As these results were suggestive of myoepithelial localization in both glands, we compared AQP8 localization with the localization of smooth muscle actin, a myoepithelial marker. We found that AQP8 and smooth muscle actin colocalized in both the rat SMG and PG, providing additional strong support for a myoepithelial localization of AQP8. Thus, in agreement with an earlier report by other investigators (Elkjaer et al., Am J Physiol Renal Physiol 281:F1047-F1057, 2001), we report that AQP8 is expressed in the myoepithelial cells, but not in the acinar cells, of both the rat SMG and PG.

In vitro cell death induced by irradiation and chemicals relevant for dental applications; dose-response and potentiation effects.

Resin-based dental materials polymerized using blue light are frequently used in dental practice and may come in contact with the oral mucosa. Remnants from oral hygiene product ingredients, such as sodium lauryl sulfate (SLS), add to the chemical exposure of the mucosa. The aim of the present in vitro study was to elucidate the cytotoxic effects in terms of apoptosis and necrosis after exposures to combinations of an adhesive (0.5% and 0.6%), SLS (concentration range 0.0025%-0.0075%), and irradiation from a dental curing lamp (radiant exposure of 8 J cm(-2)). The test system chosen was rat submandibular salivary gland acinar cells, and the cytotoxic effects were measured by fluorescence microscopy and flow cytometry methods. Cytotoxicity was observed as a result of irradiation. The most pronounced cytotoxic effects were seen in cells exposed to a combination of adhesive and SLS compared with those exposed to either agent alone. Necrosis was the dominating form of cell death for all exposures, except for the highest concentration of SLS. Apoptosis was dose-dependent on SLS in the rat submandibular acinar cells. Cytotoxic considerations of dental materials should include contributions from irradiation and other chemicals that might be present in the oral cavity.

Androgen Regulation of SMR2 Gene Expression in Rat Submandibular Gland: Evidence for a Graded but not a Binary Response

Expression of SMR2, a member of the gene family encoding salivary glutamine/glutamic acid-rich proteins, is regulated by androgens in rat submandibular gland acinar cells. To further characterize SMR2 regulation, we analyzed SMR2 expression during submandibular gland postnatal development and rat puberty at both a global and a single-cell level. Using in situ detection of mature and primary SMR2 transcripts, we show that SMR2 expression is heterogeneous among acinar cells. However, only one cell population with various amounts of mRNAs can be defined. The number of high-expressing cells increases in males during puberty and in females up to 6 weeks of age, suggesting that some factor in addition to acinar differentiation might be important for SMR2 expression in female rats. Involvement of the beta-adrenergic system in regulating SMR2 expression was tested in rats exposed daily to isoproterenol for 4 days. Under these conditions we found an increase in SMR2 expression in female rats, associated with an increase in SMR2 mRNA levels in most acinar cells. This suggests that a signaling cascade, elicited by beta-adrenergic stimuli, might act in concert with androgens to regulate SMR2 expression.

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).

Activation of phospholipase D-2 by P2X7 agonists in rat submandibular gland acini

Exogenous ATP stimulated phospholipase D (PLD), but not sphingomyelinase in rat submandibular gland (SMG) acini. PLD activation was dependent upon extracellular Ca(2+) and did not involve intracellular Ca(2+) mobilization or phosphoinositide-specific phospholipase C activation. ATP-stimulated PLD was attenuated by inhibition or downregulation of protein kinase C (PKC). PLD activation was fully blocked by the cytosolic phospholipase A(2) (PLA(2)) inhibitor ONO-RS-082 and partially attenuated by the selective Ca(2+)-dependent cytosolic PLA(2) inhibitor, arachidonyl trifluoromethylketone (AACOCF(3)), or by bromoenol lactone, an inhibitor of Ca(2+)-independent cytosolic PLA(2). Magnesium, which decreases the concentration of ATP(4-), and nickel, which blocks nonspecific cation channels coupled to purinergic receptors, inhibited PLD activation by ATP. Using reverse transcription-polymerase chain reaction and Northern blotting techniques, we demonstrated that the PLD isoform stimulated by ATP was PLD-2. Among various ATP analogs, only the P2Z/P2X(7) purinergic receptor agonist benzoyl-benzoyl ATP stimulated PLD-2. The response to ATP was inhibited by the nonselective P2X purinergic antagonist suramin and by oxidized ATP, a potent P2Z/P2X(7) receptor antagonist. It is concluded that in rat SMG acinar cells, PLD-2 is upregulated by exogenous ATP through a mechanism involving Ca(2+) influx, cytosolic PLA(2), and PKC. Also, the data suggest an involvement of P2X(7) receptors in PLD-2 stimulation by ATP.

Swelling-induced Ca2+ release from intracellular calcium stores in rat submandibular gland acinar cells.

The effects of osmotically-induced cell swelling on cytoplasmic free Ca2+ concentration ([Ca2+]i) were studied in acinar cells from rat submandibular gland using microspectrofluorimetry. Video-imaging techniques were also used to measure cell volume. Hypotonic stress (78% control tonicity) caused rapid cell swelling reaching a maximum relative volume of 1.78 +/- 0.05 (n = 5) compared to control. This swelling was followed by regulatory volume decrease, since relative cell volume decreased significantly to 1.61 +/- 0.08 (n = 5) after 10 min exposure to hypotonic medium. Osmotically induced cell swelling evoked by medium of either 78% or 66% tonicity caused a biphasic increase of [Ca2+]i. The rapid phase of this increase in [Ca2+]i was due to release of Ca2 + from intracellular stores, since it was also observed in cells bathed in Ca2+-free solution. The peak increase of [Ca2+]i induced by cell swelling was 3.40 +/- 0.49 (Fura-2 F340/F380 fluorescence ratio, n = 11) and 3.17 +/- 0.43 (n = 17) in the presence and the absence of extracellular Ca2+, respectively, corresponding to an absolute [Ca2+]i of around 1 microm. We found that around two-thirds of cells tested still showed some swelling-induced Ca2+ release (SICR) even after maximal concentrations (10(-5) M - 10(-4) M) of carbachol had been applied to empty agonist-sensitive intracellular Ca2+ stores. This result was confirmed and extended using thapsigargin to deplete intracellular Ca2+ pools. Hypotonic shock still raised [Ca2+]i in cells pretreated with thapsigargin, confirming that at least some SICR occurred from agonist-insensitive stores. Furthermore, SICR was largely inhibited by pretreatment of cells with carbonyl cyanide m-cholorophenyl hydrazone (CCCP) or ruthenium red, inhibitors of mitochondrial Ca2+ uptake. Our results suggest that the increase in [Ca2+]i, which underlies regulatory volume decrease in submandibular acinar cells, results from release of Ca2+ from both agonist-sensitive and mitochondrial Ca2+ stores.

Evidence that aquaporin-8 is located in the basolateral membrane of rat submandibular gland acinar cells

It is possible that, during primary saliva formation, aquaporins (AQPs) facilitate transcellular water flow across acinar cells to the lumina of salivary glands. In the rat submandibular gland (rSMG) AQP5 is localized in the apical membranes of acinar cells. The presence of a basolateral AQP in the same cell type has not been reported. We have therefore used immunofluorescence confocal microscopy to determine the subcellular localization of a newly discovered aquaporin, AQP8, in rSMG epithelial cells. The antibodies we used were made against the amino- or carboxyl-terminus (anti-rAQP8NT and anti-rAQP8CT, respectively) of an AQP8 cloned from rat pancreas and liver (rAQP8). Two lines of evidence suggest that both antibodies are suitable for immunolocalization studies. First, results of immunofluorescence confocal microscopy studies show that both antibodies bind to the plasma membranes of 293 cells infected with an adenovirus encoding rAQP8. Second, results of immunoblots of membranes from infected cells suggest that both antibodies bind to glycosylated and non-glycosylated forms of rAQP8. When tested in frozen sections of rSMG, we could not detect the binding of anti-rAQP8NT to any membranes. In contrast, anti-rAQP8CT binds to the basolateral membranes of acinar (but not ductal) epithelia, suggesting that rAQP8 resides in the basolateral membranes of acinar cells. Lack of anti-rAQPNT binding to basolateral membranes suggests that this epitope is not available in the membranes. Our evidence for the basolateral localization of rAQP8 in acinar cells, coupled with previous findings that AQP5 is localized apically in the same cells, raises the possibility that water crosses the acinar epithelium through these channels during primary saliva formation.

Changes in calcium concentrations in subcellular compartments of rat submandibular gland acinar cells induced by cholinergic stimulation.

The effects of cholinergic and alpha-adrenergic stimulation on calcium concentrations in rat submandibular gland acinar cells were evaluated at the subcellular level by electron probe X-ray microanalysis, and the subcellular distribution of receptors for inositol 1,4,5-trisphosphate (IP(3)Rs) was investigated by electron microscopic immunohistochemistry. For measurement of calcium concentrations, experiments were carried out both in vivo and in vitro. In in vivo experiments, fragments of glands were removed and slam-frozen 3 min after intraperitoneal injection of an agonist. In in vitro experiments, the gland tissue was enzymatically dispersed, treated with an agonist in the presence or absence of extracellular Ca(2+), and slam-frozen. The frozen specimens were cut into ultrathin cryosections, which were then freeze dried. Spectra were collected from secretory granules, the perinuclear cytoplasm containing endoplasmic reticulum (ER), and the nucleus of the acinar cells. A clear decrease in calcium content in secretory granules was observed after cholinergic stimulation both in vivo and in vitro in the presence or absence of extracellular Ca(2+). In the presence of extracellular Ca(2+), cholinergic stimulation following alpha-adrenergic stimulation reduced the calcium content in secretory granules to the same extent as cholinergic stimulation alone. No significant changes in the calcium content of the perinuclear cytoplasm and the nucleus were noticed after cholinergic stimulation. alpha-adrenergic stimulation did not significantly affect the calcium concentrations in any of the three compartments studied. IP(3)Rs were localized to ER lamellae, but not to secretory granule membranes or to nuclear membranes. Our findings suggest that: (1) in rat submandibular acini, Ca(2+) can be released from secretory granules by exposure to cholinergic, but not alpha-adrenergic, stimuli, (2) there are, however, no IP(3)Rs present in the granular membrane and the trigger for the Ca(2+) release remains under study, and (3) the response to either type of stimulus does not involve changes in the Ca(2+) content of the nucleus or of the perinuclear cytoplasm/ER, despite the presence of IP(3)Rs in the latter structure. This may be the result of limitation in the technique used, which cannot measure changes in the proper compartment of the ER.

Regulated permeability of tight junctions by E-cadherin in rat submandibular gland acini.

We examined the relationship between E-cadherin (E-CD), protein kinase C (PKC) and assembly of the tight junction (Tj) in rat submandibular gland acinar cells. The junctional complex of the acinar cells was double-labeled with anti-ZO-1 antibody and anti-E-CD antibody. When Ringer's solution was intraductally injected into the main duct, ZO-1 labels were highly concentrated at the Tj zone, and the adherens junction (Aj) was exclusively labeled by E-CD. In addition, the Tj was impermeable to microperoxidase. Neither intraductal injection of anti-E-CD antibody solution nor infusion of carbachol produced labels for ZO-1 at the Tj zone, although these proteins were occasionally intermixed at the Aj zone. In these cases, the Tj was permeable to microperoxidase. Intraductal injection of anti-E-CD antibody solution with PKC agonist, resulted in a reduction of E-CD labels in the Aj zone, while the ZO-1 was labeled exclusively in the Tj zone. In this case, Tj was impermeable to microperoxidase. These results suggest that E-CD plays a major role in mediating intercellular physical adhesion, and that PKC may be active in signaling the pathway activated by E-CD-mediated cell-cell adhesion.

Involvement of Microtubules and Globular Structures in the Membrane Traffic of Clathrin-Coated Vesicles During Membrane Retrieval in Rat Submandibular Gland Acinar Cells.

Involvement of Microtubules and Globular Structures in the Membrane Traffic of Clathrin-Coated Vesicles During Membrane Retrieval in Rat Submandibular Gland Acinar Cells.

Membrane-specific Regulation of Cl− Channels by Purinergic Receptors in Rat Submandibular Gland Acinar and Duct Cells

Measurement of [Cl-]i and the Cl- current in the rat salivary submandibular gland (SMG) acinar and duct cells was used to evaluate the role of Cl- channels in the regulation of [Cl-]i during purinergic stimulation. Under resting conditions [Cl-]i averaged 56 +/- 8 and 26 +/- 7 mM in acinar and duct cells, respectively. In both cells, stimulation with 1 mM ATP resulted in Cl- efflux and subsequent influx. Inhibition of NaKCl2 cotransport had no effect on [Cl-]i changes in duct cells and inhibited only about 50% of Cl- uptake in acinar cells. Accordingly, low levels of expression of NaKCl2 cotransporter protein were found in duct cells. Acinar cells expressed high levels of the cotransporter. Measurement of Cl- current under selective conditions revealed that acinar and duct cells express at least five distinct Cl- channels; a ClCO-like, volume-sensitive, inward rectifying, Ca2+-activated and CFTR-like Cl- currents. ATP acting on both cell types activated at least two channels, the Ca2+-activated Cl- channel and a Ca2+-independent glibenclamide-sensitive Cl--current, possibly cystic fibrosis transmembrane regulator (CFTR). Of the many nucleotides tested only 2'-3'-benzoylbenzoyl (Bz)-ATP and UTP activated Cl- channels in SMG cells. Despite their relative potency in increasing [Ca2+]i, BzATP in both SMG cell types largely activated the Ca2+-independent, glibenclamide-sensitive Cl- current, whereas UTP activated only the Ca2+-dependent Cl- current. We interpret this to suggest that BzATP and UTP largely activate Cl- channels residing in the membrane expressing the receptor for the active nucleotide. The present studies reveal a potentially new mechanism for transcellular Cl- transport in a CFTR-expressing tissue, the SMG. Coordinated action of the P2z (luminal) and P2u (basolateral) receptors can mediate part of the transcellular Cl- transport by acinar and duct cells to determine the final electrolyte composition of salivary fluid.

Characterization and Localization of P2 Receptors in Rat Submandibular Gland Acinar and Duct Cells

[Ca2+]i and the Cl- current were measured in isolated submandibular gland acinar and duct cells to characterize and localize the purinergic receptors expressed in these cells. In both cell types 2'-3'-benzoylbenzoyl (Bz)-ATP and ATP increased [Ca2+]i mainly by activation of Ca2+ influx. UTP had only minimal effect on [Ca2+]i at concentrations between 0.1 and 1 mM. However, a whole cell current recording showed that all nucleotides effectively activated Cl- currents. Inhibition of signal transduction through G proteins by guanyl-5'-beta-thiophosphate revealed that the effect of ATP on Cl- current was mediated in part by activation of a G protein-coupled and in part by a G protein-independent receptor. BzATP activated exclusively the G protein-independent portion, whereas UTP activated only the G protein-dependent portion of the Cl- current. Measurement of [Ca2+]i in the microperfused duct showed that ATP stimulated a [Ca2+]i increase when applied to the luminal or the basolateral sides. BzATP increased [Ca2+]i only when applied to the luminal side, whereas UTP at 100 microM increased -Ca2+-i only when applied to the basolateral side. The combined results suggest that duct and possibly acinar cells express P2z receptors in the luminal and P2u receptors in the basolateral membrane.

Effects of cholinergic and alpha-adrenergic agonists on the monovalent ion content of rat submandibular gland acinar cells studied by X-ray microanalysis.

The effects of cholinergic and alpha-adrenergic stimulation (in vivo and in vitro) on the monovalent ion content of rat submandibular gland acinar cells were evaluated at the subcellular level by X-ray microanalysis. Fragments of glands or enzymatically dispersed acini were slam-frozen and cut into ultrathin cryosections. Spectra were collected from secretory granules, nucleus, the basal cytoplasm containing endoplasmic reticulum and the apical cytoplasm identified between secretory granules. No significant changes in Na and Cl content were observed after the isolation of acini, but the K concentration decreased compared with cells from in situ glands. The Cl and K content in all four compartments studied decreased significantly after cholinergic stimulation both in vivo and in vitro but in a more restricted fashion after alpha-adrenergic stimulation. Our findings indicate that: (1) the physiological mechanisms regulating the monovalent ion composition of submandibular cells are relatively well preserved in isolated acinar cells; (2) the results from in vivo experiments are in good agreement with those from in vitro experiments; and (3) the effects of cholinergic and alpha-adrenergic stimulation on the K+ and Cl- efflux at the subcellular level are similar but the response is generally less with alpha-adrenergic stimulation.

Development and characterization of SV40 immortalized rat submandibular acinar cell lines

Rat submandibular salivary gland acinar cells were transfected by CaPO4 precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 27 clonal cell lines, two were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the beta-adrenergic agonist, isoproterenol, vasoactive intestinal peptide, and prostaglandin E1 were effective activators of intracellular cyclic AMP production. Epinephrine, norepinephrine, phenylephrine, acetylcholine, and P2U-purinoceptor agonists were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without effect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express glutamine/glutamic acid-rich proteins, a submandibular acinar cell specific secretory protein family. Electron microscopic evaluation documented the maintenance of tripartite junctional complexes, cellular polarization, and the presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 25 h.

Depletion of ryanodine-sensitive Ca2+ store activates Ca2+ entry in rat submandibular gland acinar cells.

The existence of ryanodine-sensitive Ca2+ stores and their role in the Ca2+ entry mechanism were examined in the rat submandibular gland acinar cells, using the microfluorimetry of intracellular Ca2+ concentration ([Ca2+]i). In the presence of thapsigargin, a Ca(2+)-ATPase inhibitor of inositol (1, 4, 5) triphosphate (InsP3)-sensitive Ca2+ stores, caffeine caused an increase in [Ca2+]i, which was inhibited by treatment with ryanodine (a ligand to the Ca(2+)-induced Ca2+ release channels). In the cells treated with ryanodine, 1 mM Ca2+ addition to a Ca(2+)-free solution caused a marked increase in [Ca2+]i, which was eliminated by application of Ni2+ or SK & F 96365, suggesting a Ca2+ entry triggered by ryanodine. The maximal change in the net increase in [Ca2+]i caused by the ryanodine-coupled Ca2+ entry, was 104.0 +/- 16.0 nM, which intense was caused by 10 microM ryanodine. Emptying the InsP3-sensitive stores by treatment with thapsigargin also caused Ca2+ entry, which maximally changed [Ca2+]i by 349.6 +/- 15.1 nM. Ten mumol/liter ryanodine was confirmed to cause a release of 45Ca2+ from the parotidic microsomal fraction enriched in endopalsmic reticulum. We propose that ryanodine-sensitive Ca2+ stores are present in rat submandibular gland acinar cells. We further propose that release of Ca2+ from the ryanodine-sensitive stores, which means eventually depletion of the ryanodine-sensitive Ca2+ stores, can activate the Ca2+ entry. The ability for Ca2+ entry coupled with the ryanodine-sensitive Ca2+ stores seems to be about 30% of the ability for Ca2+ entry coupled with the thapsigargin-sensitive Ca2+ stores.

Changes in glycoconjugates in rat submandibular gland after chronic treatment with reserpine and isoproterenol.

Chronic treatment of rats with reserpine, isoproterenol, or a combination of these two agents has been suggested as a means to produce an experimental animal model for the chronic exocrinopathy cystic fibrosis. The effect of these treatments on glycoconjugate distribution in rat submandibular gland acinar cells was investigated by quantitative lectin cytochemistry. Significant changes in wheat-germ agglutinin (WGA), soy bean agglutinin (SBA) and concanavalin A (Con A) binding sites in the mucus granules were observed, but peanut agglutinin (PNA) binding was not significantly affected. The quantitative changes in glycoconjugates in the acinar cells of the submandibular gland could be a possible explanation for the increased binding of calcium by the intracellular mucus noted in previous studies on these animal models.

Muscarinic, α1 and peptidergic agonists stimulate phosphoinositide hydrolysis and regulate mucin secretion in rat submandibular gland cells

Three classes of agonists associated with Ca2+-mobilization--alpha 1-adrenergic (methoxamine), muscarinic (carbachol) and peptidergic (substance P, SP)--significantly stimulated the secretion of mucin from enzymatically-dispersed rat submandibular gland acinar cells. The same three secretagogues also caused the hydrolysis of membrane inositol phospholipids, resulting in elevated cellular levels of inositol phosphates, particularly inositol 1,4,5-trisphosphate (IP3). Exogenous IP3 elicited the dose-dependent release of mucin in dispersed cells suggesting that agonist-generated endogenous IP3 may provoke a secretory response. IP3 and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in combination, stimulated an additive secretion of mucin in the model. The potential use of these two agents as specific probes of the IP3- and diacylglycerol-associated legs of the polyphosphoinositide (PPI) breakdown pathway is indicated. Although all three agonists shared a common action in stimulating PPI hydrolysis, their effects on the beta-adrenergic mucosecretory response were inconsistent. A brief preincubation of cells with carbachol or SP significantly reduced the subsequent isoproterenol (IPR)-provoked secretion of mucin, whereas methoxamine plus IPR stimulated an additive response. The mechanisms underlying these opposite effects are not known. Failure of IP3 or TPA to modify IPR responses suggests that modulation of the beta response may operate at a locus before the generation of diacylglycerol and IP3, possibly at the level of signal transduction. The study indicates a role for Ca2+-mobilizing agonists in controlling submandibular mucin secretion and provides evidence that receptor-linked phosphoinositide hydrolysis is an early stage in their stimulus-secretion coupling mechanism.