Ca signaling and regulation of fluid secretion in salivary gland acinar cells

Neurotransmitter stimulation of plasma membrane receptors stimulates salivary gland fluid secretion via a complex process that is determined by coordinated temporal and spatial regulation of several Ca²⁺ signaling processes as well as ion flux systems. Studies over the past four decades have demonstrated that Ca²⁺ is a critical factor in the control of salivary gland function. Importantly, critical components of this process have now been identified, including plasma membrane receptors, calcium channels, and regulatory proteins. The key event in activation of fluid secretion is an increase in intracellular [Ca²⁺] ([Ca²⁺]_i) triggered by IP₃-induced release of Ca²⁺ from ER via the IP₃R. This increase regulates the ion fluxes required to drive vectorial fluid secretion. IP₃Rs determine the site of initiation and the pattern of [Ca²⁺]_i signal in the cell. However, Ca²⁺ entry into the cell is required to sustain the elevation of [Ca²⁺]_i and fluid secretion. This Ca²⁺ influx pathway, store-operated calcium influx pathway (SOCE), has been studied in great detail and the regulatory mechanisms as well as key molecular components have now been identified. Orai1, TRPC1, and STIM1 are critical components of SOCE and among these, Ca²⁺ entry via TRPC1 is a major determinant of fluid secretion. The receptor-evoked Ca²⁺ signal in salivary gland acinar cells is unique in that it starts at the apical pole and then rapidly increases across the cell. The basis for the polarized Ca²⁺ signal can be ascribed to the polarized arrangement of the Ca²⁺ channels, transporters, and signaling proteins. Distinct localization of these proteins in the cell suggests compartmentalization of Ca²⁺ signals during regulation of fluid secretion. This chapter will discuss new concepts and findings regarding the polarization and control of Ca²⁺ signals in the regulation of fluid secretion.     

ATP-induced calcium signalling in acinar cells of the submandibular salivary gland

To study changes in the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and the total amount of calcium in cells, we used, respectively, the fluorescent dye fura 2/AM and the metallochrome dye arsenazo III. The total amount of calcium in acinar cells after their incubation in calcium-free ATP-containing extracellular solution decreased. The action of ATP induced a dose-dependent increase in the [Ca²⁺]_i; the EC₅₀ was, on average, 130 ± ± 36 μM. Calcium transients induced by ATP demonstrated no desensitization. Against the background of a blocker of ionotropic P2X receptors, pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid, we observed a decrease in the ATP-induced calcium transients by 72%. In addition, these transients were reduced by 65% in the calcium-free milieu, while after thapsigargin-induced exhaustion of the endoplasmic reticulum store they disappeared. This is indicative of the involvement of metabotropic P2Y receptors in the formation of the above calcium transients. Therefore, P2X and P2Y receptors participate in ATP-induced calcium signalling in acinar cells of the submandibular salivary gland; activation of these channels results in a rise in the [Ca²⁺]_i. The P2X receptors to a higher extent contribute to the formation of calcium signals; the P2Y-determined increase in the [Ca²⁺]_i is smaller (equal to about 35%). Therefore, the functionally active ligand-operated ionotropic P2Y receptors and metabotropic G protein-related P2Y receptors do exist in acinar cells of the submandibular salivary gland and play an important role in the control of functioning of this gland. Neirofiziologiya/Neurophysiology, Vol. 37, Nos. 5/6, pp. 395–402, September–December, 2005.     

Insulin-stimulated protein synthesis in submandibular acinar cells: interactions with adrenergic and cholinergic agonists

The effects of insulin and secretory agonists on amino acid incorporation into submandibular gland proteins were studied using isolated acinar cell aggregates. Insulin stimulated the incorporation of 3H-leucine into TCA-precipitable proteins in a rapid, dose-dependent manner (half-maximal response at 1 nM). Isoproterenol, a beta-adrenergic agonist, also stimulated amino acid incorporation, and this effect was mimicked by both dibutyryl cAMP and IBMX, a phosphodiesterase inhibitor. Although insulin further stimulated incorporation in the presence of isoproterenol and IBMX, no additional increase in the rate of synthesis was observed after stimulation by dibutyryl cAMP. High concentrations of carbamylcholine, a cholinergic agonist, inhibited both basal and insulin-stimulated incorporation. At low concentrations, however, carbamylcholine stimulated synthesis, and the effects of insulin and carbamylcholine were additive. A23187, a calcium ionophore, also inhibited 3H-leucine incorporation and insulin stimulation, but in contrast to carbamylcholine, low concentrations of A23187 neither inhibited nor enhanced the rate of synthesis. Thus, protein synthesis in the rat submandibular gland is regulated by both insulin and neurotransmitters. Whereas beta-adrenergic stimulation appears to be mediated through cAMP, the intracellular signals mediating the actions of insulin and cholinergic agonists remain to be elucidated.     

Physiological and morphological evidence for coupling in mouse salivary gland acinar cells

Three experimental techniques were employed to examine coupling between acinar cells of the mouse salivary gland. Passage of DC current pulses via intracellular microelectrodes between neighboring cells showed that small ions could be directly passed from one cell to another. Intracellular iontophoresis of the dye Lucifer Yellow CH into a single cell indicated that small molecules could spread by means of intercellular cytoplasmic bridges througout an acinus and, occasionally, into cells of adjacent acini. Freeze-fracture replicas of acinar cell membranes indicated the presence of gap junctions which were correlated with both electrical and dye coupling experiments. Suggestions are made for the function of direct intercellular exchange in salivary secretory cells. The role of electrical coupling in coordination of the activity of different secretory cell types is discussed as one possible function.     

Persistent ultrastructural changes in pancreatic acinar cells induced by 4-acetylaminofluorene

Male Leeds rats were fed a diet containing 0.05% of the non-carcinogen 4-acetylaminofluorene (4-AAF) for 8–10 months. They were then returned to a normal diet and their pancreatic tissues examined by electron microscopy at intervals between 2 and 12 months after the end of 4-AAF treatment. 4-AAF was found to induce a persistent alteration in the morphology of the granular endoplasmic reticulum, involving distortion and dilatation of the cisternae. In some respects this lesion resembles that which is induced by the carcinogenic isomer, 2-acetylaminofluorene (2-AAF).     

Immunocytochemical localization of amylase in gerbil salivary gland acinar cells processed by rapid freezing and freeze-substitution fixation

We examined the immunocytochemical localization of amylase in cryofixed serous acinar cells of gerbil major salivary glands by indirect immunostaining, using anti-gerbil parotid amylase antibody and protein A-gold complex. Fresh tissue blocks were quickly frozen by the metal-contact method, using liquid helium, and were freeze-substituted with either osmium-acetone solution or glutaraldehyde-containing acetone. They were then embedded in an epoxy resin mixture which was polymerized at 60 degrees C. Some tissue blocks substituted with aldehyde-acetone solution were embedded in Lowicryl K4M, polymerized at -30 degrees C. Thin sections of epoxy resin-embedded materials were treated with an oxidizing agent before immunostaining. The labeling density on the materials processed by various protocols for preparatory procedures was quantitatively compared to examine the usefulness of application of cryofixation to immunocytochemistry. The central dense core of heterogeneous secretory granules in the serous acinar cells of the parotid and sublingual glands was heavily labeled with immunogold, regardless of substitution media and embedding resins employed. The immunolabeling pattern clearly distinguished between the dense core and the surrounding matrix. Labeling density in the cryofixed materials was about 1.5 times greater than in those processed by conventional chemical fixation. Seromucous secretory granules in the submandibular gland acinar cells were only faintly labeled. The results obtained indicate that application of immunostaining to quick-frozen, substitution-fixed tissues is useful for high-resolution immunocytochemistry.     

Activation of PKC is sufficient to induce an apoptotic program in salivary gland acinar cells

Accumulating evidence suggests that specific isoforms of PKC may function to promote apoptosis. We show here that activation of the conventional and novel isoforms of PKC with 12-O-tetradecanoyl phorbol-13- ester (TPA) induces apoptosis in salivary acinar cells as indicated by DNA fragmentation and activation of caspase-3. TPA-induced DNA fragmentation, caspase-3 activation, and morphologic indicators of apoptosis, can be enhanced by pretreatment of cells with the calpain inhibitor, calpeptin, prior to the addition of TPA. Analysis of PKC isoform expression by immunoblot shows that TPA-induced downregulation of PKC alpha and PKC delta is delayed in cells pre-treated with calpeptin, and that this correlates with an increase of these isoforms in the membrane fraction of cells. TPA-induced apoptosis is accompanied by biphasic activation of the c-jun-N-terminal kinase (JNK) pathway and inactivation of the extracellular regulated kinase (ERK) pathway. Expression of constitutively activated PKC alpha or PKC delta, but not kinase negative mutants of these isoforms, or constitutively activated PKC epsilon, induces apoptosis in salivary acinar cells, suggesting a role for these isoforms in TPA-induced apoptosis. These studies demonstrate that activation of PKC is sufficient for initiation of an apoptotic program in salivary acinar cells. Cell Death and Differentiation (2000) 7, 1200 - 1209.     

Amino acid induced changes of puffing patterns in Chironomus salivary gland chromosomes

Effects of amino acids on puffing in salivary gland chromosomes of Chironomus thummi are described. All twenty naturally occurring amino acids except tyrosine were tested. Of these, only tryptophan and phenylalanine proved effective. Both induced puffs at the same seven loci: Ia3/4, Ie3, If4, Ig1, Ig2, IIId1 and IIId1/2. The induction is energy dependent and requires concomitant RNA synthesis. Amino acid induced puffs incorporate ³H-uridine into acid insoluble material. At least one monovalent plus one divalent cation has to be present along with the amino acid in the incubation medium for puff induction.     

Protein kinase C delta is essential for etoposide-induced apoptosis in salivary gland acinar cells.

We have previously shown that parotid C5 salivary acinar cells undergo apoptosis in response to etoposide treatment as indicated by alterations in cell morphology, caspase-3 activation, DNA fragmentation, sustained activation of c-Jun N-terminal kinase, and inactivation of extracellular regulated kinases 1 and 2. Here we report that apoptosis results in the caspase-dependent cleavage of protein kinase C-delta (PKCdelta) to a 40-kDa fragment, the appearance of which correlates with a 9-fold increase in PKCdelta activity. To understand the function of activated PKCdelta in apoptosis, we have used the PKCdelta-specific inhibitor, rottlerin. Pretreatment of parotid C5 cells with rottlerin prior to the addition of etoposide blocks the appearance of the apoptotic morphology, the sustained activation of c-Jun N-terminal kinase, and inactivation of extracellular regulated kinases 1 and 2. Inhibition of PKCdelta also partially inhibits caspase-3 activation and DNA fragmentation. Immunoblot analysis shows that the PKCdelta cleavage product does not accumulate in parotid C5 cells treated with rottlerin and etoposide together, suggesting that the catalytic activity of PKCdelta may be required for cleavage. PKCalpha and PKCbeta1 activities also increase during etoposide-induced apoptosis. Inhibition of these two isoforms with G?6976 slightly suppresses the apoptotic morphology, caspase-3 activation, and DNA fragmentation, but has no effect on the sustained activation of c-Jun N-terminal kinase or inactivation of extracellular regulated kinase 1 and 2. These data demonstrate that activation of PKCdelta is an integral and essential part of the apoptotic program in parotid C5 cells and that specific activated isoforms of PKC may have distinct functions in cell death.     

Human salivary gland acinar cells spontaneously form three-dimensional structures and change the protein expression patterns

Applying tissue engineering principles to design an auto-secretory device is a potential solution for patients suffering loss of salivary gland function. However, the largest challenge in implementing this solution is the primary culture of human salivary gland cells, because the cells are highly differentiated and difficult to expand in vitro. This situation leads to the lack of reports on the in vitro cell biology and physiology of human salivary gland cells. This study used a low-calcium culture system to selectively cultivate human parotid gland acinar (PGAC) cells from tissues with high purity in cell composition. This condition enables PGAC cells to continuously proliferate and retain the phenotypes of epithelial acinar cells to express secreting products (α-amylase) and function-related proteins (aquaporin-3, aquaporin-5, and ZO-1). Notably, when the cells reached confluence, three-dimensional (3D) cell aggregates were observed in crowded regions. These self-formed cell spheres were termed post-confluence structures (PCSs). Unexpectedly, despite being cultured in the same media, cells in PCSs exhibited higher expression levels and different expression patterns of function-related proteins compared to the two-dimensional (2D) cells. Translocation of aquoporin-3 from cytosolic to alongside the cell boundaries, and of ZO-1 molecules to the boundary of the PCSs were also observed. These observations suggest that when PGAC cells cultured on the 2D substrate would form PCSs without the help of 3D scaffolds and retain certain differentiation and polarity. This phenomenon implies that it is possible to introduce 2D substrates instead of 3D scaffolds into artificial salivary gland tissue engineering.     

Membrane potential perturbations induced in tissue cells by pulsed electric fields

Pulsed electric fields directly influence the electrophysiology of tissue cells by transiently perturbing their transmembrane potential. To determine the magnitude and time course of this interaction, electrotonic cable theory was used to calculate the membrane potential perturbations induced in tissue cells by a spatially uniform, pulsed electric field. Analytic solutions were obtained that predict shifts in membrane potential along the length of cells as a function of time in response to an electrical pulse. For elongated tissue cells, or groups of tissue cells that are coupled electrotonically by gap junctions, significant hyperpolarizations and depolarizations can result from millisecond applications of electric fields with strengths on the order of 10–100 mV/cm. The results illustrate the importance of considering cellular cable parameters in assessing the effects of transient electric fields on biological systems, as well as in predicting the efficacy of pulsed electric fields in medical treatments. © 1995 Wiley-Liss, Inc.     

Caffeine-induced calcium release from the endoplasmic reticulum of acinar cells of the submandibular salivary gland

Ryanodine receptors (RyRs) play a key role in the generalization and spreading of calcium waves in excitable cells; however, the question of the existence of functionally active RyRs in nonexcitable cells demonstrating the capacity for exocytosis (e.g., salivary gland acini) remains open. We studied changes in the total amount of calcium stored in the endoplasmic reticulum (ER) of acinar cells of the submandibular salivary gland of rats and changes in the concentration of ionized Ca²⁺ inside the ER ([Ca²⁺]_ER) using, respectively, a metallochrome dye, arsenazo III, and a low-affinity fluorescent dye, mag-fura 2/AM. In permeabilized cells, caffeine caused dose-dependent decreases in the total amount of calcium and concentration of ionized calcium. The effective concentration of caffeine providing a 50% drop in the [Ca²⁺]_ER (EC₅₀) was, on average, 7.3 ± 1.1 mM. The caffeine-induced drop in the [Ca²⁺]^ER was insensitive to heparin; in addition, it was blocked by high concentrations (100 μM) of ryanodine, potentiated by ryanodine applied in mild concentrations (10 μM), and also demonstrated a bell-shaped dependence on the concentration of cytoplasmic Ca²⁺. Such peculiarities are typical characteristics of the RyR-mediated reaction. Therefore, functional RyRs whose activation results in a transient release of calcium from the ER are present in acinar cells of the submandibular salivary gland. Neirofiziologiya/Neurophysiology, Vol. 39, No. 2, pp. 107–112, March–April, 2007.     

Tracheal submucosal gland serous cells stimulated in vitro with adrenergic and cholinergic agonists A morphometric study

Summary A morphometric analysis was made of alterations in serous cell structure induced by adrenergic and cholinergic agonists. Ferret tracheal rings were exposed for 30 min in vitro to one of the following agonists: phenylephrine, terbutaline, or methacholine (all at 10^–5 M). Controls were incubated similarly in medium containing no drugs or medium containing both the agonist and an excess of the appropriate antagonist (phentolamine, propranolol or atropine, all at 10^–4 M).Electron microscopic observation and stereological analysis of the incubated samples revealed that the volume density of serous cell granules in controls (0.30 ± 0.02, mean ± SE, n = 4) was significantly reduced by phenylephrine (0.19 ± 0.03, n = 4) and methacholine (0.17 ± 0.01, n = 4), but not by terbutaline (0.27 ± 0.04, n = 4). The presence of antagonists in the medium prevented the observed changes (phenylephrine/phentolamine: 0.29 ± 0.03, n = 3 and methacholine/atropine: 0.33 ± 0.06, n = 3). In addition, the volume density of intracellular vacuoles in controls (0.02 ± 0.005, n = 4) was increased in response to methacholine stimulation (0.12 + 0.05, n = 4), but not in response to the other agonists. This effect was blocked by atropine (0.01 ± 0.00, n = 3).We conclude that serous-cell granules are discharged by both alpha-adrenergic and cholinergic, but not beta-adrenergic stimulation. In addition, cholinergic stimulation evokes the formation of intracellular vacuoles, a possible indication of active ion and water transport.     

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

 The distribution of anti-Müllerian hormone (AMH) and laminin (Ln) α5 chain in differentiating rat testis and ovary were studied by immunohistochemistry. In the incipient embryonic male gonad a weak reaction for Ln α5 chain, but not for AMH, was detected. With further prenatal development, Ln α5 chain rapidly disappeared from the basement membrane (BM) of the incipient testicular cords in parallel with the appearance of AMH in the Sertoli cells. After birth, Ln α5 chain reappeared in the BMs of the cords with the decline and disappearance of AMH from the respective Sertoli cells. In the corresponding stages of the ovary, Ln α5 chain was present in the BM of the prenatal gonadal cords and in postnatal primordial follicles. The cells of those epithelia were negative for AMH. With the growth of the follicles, Ln α5 chain disappeared from the BM when AMH appeared in the epithelial follicular cells. The present results show that male and female gonadal epithelia negative for Ln α5 chain were positive for AMH, and that epithelia positive for Ln α5 chain were negative for AMH. Thus, epithelial Ln α5 chain and AMH as a product of the same cell seemed to exclude each other. The results require an explanation why Ln α5 chain has to be excluded from the BM of the epithelia during the secretion of AMH chain through the basal cell membrane to the surrounding tissues where it executes its important biological functions. These observations suggest a hypothesis that the production of both components is regulated by the same gene and factor system. Accepted: 14 January 1999