Ultrastructural and immunocytochemical investigation of ecdysteroid secretion by the prothoracic gland of the waxmoth Galleria mellonella

Summary The formation and secretion of ecdysteroid by the prothoracic gland cells of Galleria mellonella (Insecta, Lepidoptera) were investigated electron microscopically and immunocytochemically. The moulting hormone ecdysone becomes first evident in vesicles and tubules of the smooth endoplasmic reticulum (SER). The SER forms secretory granules in which ecdysone was shown immunocytochemically. The Golgi apparatus seems not to be directly involved in ecdysone secretion. The secretory granules are released from the cells by exocytosis.Supported by the Sächsische Akademie der Wissenschaften zu LeipzigThe author is grateful to Mrs. Angelika Schmidt for her excellent assistance     

Maturation-related changes in mass and elemental contents of secretory granules as measured by electron-microprobe

Summary The relationship between granule density, protein content, and Ca and S contents were studied in two secretory granule fractions, from parotid glands of the rat, previously shown to constitute different stages in granule maturation. The density of the lighter fraction was between 1.133 and 1.142 g/ml, while that of the heavier fraction was greater than 1.142 g/ml. The mean protein content of the denser granules was 12% greater than that of the lighter granules (P<0.03), while the dry-mass elemental concentrations in the two granule fractions were unchanged. These results indicate that protein is added to granules during the maturation process (presumably by vesicular traffic), and that the resulting increase in granule density is not driven simply by decrease in water content and/or increased concentrations of inorganic Ca or S in the granules. The elemental concentration values also indicate that the diffusible elements permeate the granule membrane during the fractionation procedures.     

Difference in distribution of membrane proteins between low- and high-density secretory granules in parotid acinar cells

Secretory granules (SGs) are considered to be generated as immature granules and to mature by condensation of their contents. In this study, SGs of parotid gland were separated into low-, medium-, and high-density granule fractions by Percoll-density gradient centrifugation, since it was proposed that the density corresponds to the degree of maturation. The observation with electron microscopy showed that granules in the three fractions were very similar. The average diameter of high-density granules was a little but significantly larger than that of low-density granules. Although the three fractions contained amylase, suggesting that they are all SGs, distribution of membrane proteins was markedly different. Syntaxin6 and VAMP4 were localized in the low-density granule fraction, while VAMP2 was concentrated in the high-density granule fraction. Immunoprecipitation with anti-syntaxin6 antibody caused coprecipitation of VAMP2 from the medium-density granule fraction without solubilization, but not from Triton X-100-solubilized fraction, while VAMP4 was coprecipitated from both fractions. Therefore, VAMP2 is present on the same granules, but is separated from syntaxin6 and VAMP4, which are expected to be removed from immature granules. These results suggest that the medium-density granules are intermediates from low- to high-density granules, and that the membrane components of SGs dynamically change by budding and fusion during maturation.     

Intracellular transport of sulfated macromolecules in parotid acinar cells

Intracellular transport of sulfated macromolecules in parotid acinar cells was investigated by electron microscopic radioautography after injection of 35S-sulfate. Ten minutes after injection radiosulfate was concentrated in the Golgi region. By 1 hr, much of the radioactive material had been transported to condensing vacuoles. These vacuoles were subsequently transformed into zymogen granules which contained almost 70% of the radioactivity 4 hrs after injection. These results indicate that, in addition to its packaging function, the Golgi apparatus in parotid acinar cells is capable of utilizing inorganic sulfate for the production of sulfated macromolecules. These molecules, following an intracellular route similar to that taken by digestive enzymes, become an integral component of zymogen granules. The possibility that sulfated macromolecules play a role in exocrine secretion by aiding in the packaging of exportable proteins is discussed.     

Isolation and partial characterization of two populations of secretory granules from rat parotid glands

Summary A method is described for the isolation of two populations of secretory granules from rat parotid glands utilizing differences in their sedimentation characteristics. The granule preparations were analyzed for homogeneity by electron microscopy and chemical analyses. The soluble contents of both types of granules were obtained by hypotonic lysis, and the proteins compared by SDS-PAGE and ion exchange-gel filtration chromatography. Both populations of secretory granules appear to have the same protein composition as that of the parotid saliva. The secretory granules with the smaller apparent buoyant density became labelled with radioactive leucine earlier than the heavier granules when a pulse of this amino acid was supplied to a gland slice system. The lighter granules appear to represent an earlier stage in maturation.     

Involvement of phospholipase D in the cAMP-regulated exocytosis of rat parotid acinar cells

The activation of beta-adrenergic receptors in rat parotid acinar cells causes intracellular cAMP elevation and appreciably stimulates the exocytotic release of amylase into saliva. The activation of Ca(2+)-mobilizing receptors also induces some exocytosis. We investigated the role of phospholipase D (PLD) in regulated exocytosis in rat parotid acinar cells. A transphosphatidylation assay detected GTPgammaS (a nonhydrolyzable analogue of GTP)-dependent PLD activity in lysates of rat parotid acinar cells, suggesting that PLD is activated by small molecular mass GTP-binding proteins. The PLD inhibitor, neomycin, suppressed cAMP-dependent exocytosis in saponin-permeabilized cells. Signaling downstream of PLD was disrupted by 1-butanol due to conversion of the PLD reaction product (phosphatidic acid) to phosphatidylbutanol. The stimulation of exocytosis by isoproterenol as well as by a Ca(2+)-mobilizing agonist (methacholine) was inhibited by 1-butanol. These results suggest that PLD is important for regulated exocytosis in rat parotid acinar cells.     

Quantitative analyses of the constituent membranes of parotid acinar cells and of the changes evident after induced exocytosis

Summary A morphometric study has been made at the EM level of Isoproterenol (IPR) induced secretion of rabbit parotid glands in vivo. Emphasis has been placed here on the membrane content of acinar cells and the changes which occur following induced degranulation. In particular it was hoped to establish whether the preservation of zymogen granule membrane as intact electron microscopically visible subunits and the subsequent reutilisation of this membrane is a plausible hypothesis from a quantitative morphological standpoint.After two hours IPR had caused >95% depletion of granules. About 1343 m²/cell of granule limiting membrane temporarily fused with the apical plasmalemma during this time and by two hours 1158 m²/cell of this had been eliminated. Only a small increase in intracellular smooth membrane area was recorded after degranulation and we find no evidence that the zymogen granule membrane is stored indefinitely as smooth membrane fragments either in the region of the Golgi apparatus or elsewhere in the cytoplasm.IPR caused changes in RER membrane area (+37.7%, 1406 m²/cell), which is a possible, but we consider implausible relocation site of granule membrane.The possible mechanism of the removal of excess apical membrane and the ultimate fate of the zymogen granule membrane is discussed.     

Unstimulated amylase secretion is proteoglycan-dependent in rat parotid acinar cells

It is well-known that amylase is secreted in response to extracellular stimulation from the acinar cells. However, amylase is also secreted without stimulation. We distinguished vesicular amylase as a newly synthesized amylase from the accumulated amylase in secretory granules by short time pulse and chased with ³⁵S-amino acid. The newly synthesized amylase was secreted without stimulation from secretory vesicles in rat parotid acinar cells. The secretion process did not include microtubules, but was related to microfilaments. p-Nitrophenyl β-xyloside, an inhibitor of proteoglycan synthesis, inhibited the newly synthesized amylase secretion. This indicated that the newly synthesized amylase was secreted from secretory vesicles, not via the constitutive-like secretory route, which includes the immature secretory granules, and that proteoglycan synthesis was required for secretory vesicle formation.     

Zymogen granules of mouse parotid acinar cells are acidified in situ in an ATP-dependent manner

Summary The mechanism for acidification of zymogen granules in acinar cells of mouse parotid gland was explored using acridine orange, lysosomotropic agents, and an inhibitor of cellular ATP production. Methylamine and monensin reversibly collapsed the pH gradient of granules without affecting cellular ATP levels. Depletion of cellular ATP with antimycin A did not collapse the pH gradient. However, recovery of acidity in the granules, after collapse of the pH gradient by methylamine, was blocked by depletion of cellular ATP. These results demonstrate that zymogen granules of parotid gland are acidic in situ and that ATP is required for acidification of the granules.     

Ultrastructural changes associated with renin secretion from the juxtaglomerular apparatus of mice

Summary Thin sections and freeze-fracture replicas were used to investigate the ultrastructural changes associated with renin secretion from the juxtaglomerular part of the afferent arteriole of male mice. Adrenalectomized animals in which renin secretion was stimulated by furosemide application and bleeding were also studied. Exocytosis of mature electron-dense granules was found in all experimental groups. Before extrusion, the region of granule facing the cell membrane changed, with vesicular and/or stacked membrane-like profiles and a small local protrusion of the granule membrane appearance of. Concomitantly, punctuate sites of fusion between the cell and granule membranes were observed. Later, unaltered amorphous, and altered membrane-like granule content was released from omega-shaped cavities into the extracellular space. In stimulated animals the alteration and extrusion of several closely apposed granules was reminiscent of compound exocytosis. Coated pits were frequently seen, suggesting specific retrieval of the former granule membrane. The collapsing silhouette of a depleted granule very rarely took the form of a saccule whose narrow membrane-bounded neck was continuous with the extracellular space.Observed were two additional events by which active and inactive renin may be released. Small electron-lucent vacuoles of undetermined origin fused with the cell membrane and, in stimulated kidneys, some epithelioid cell processes disintegrated. However, the interpretation of the related ultrastructural phenomena was uncertain.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Ca-regulated secretory granule exocytosis in pancreatic and parotid acinar cells

Protein secretion from acinar cells of the pancreas and parotid glands is controlled by G-protein coupled receptor activation and generation of the cellular messengers Ca²⁺, diacylglycerol and cAMP. Secretory granule (SG) exocytosis shares some common characteristics with nerve, neuroendocrine and endocrine cells which are regulated mainly by elevated cell Ca²⁺. However, in addition to diverse signaling pathways, acinar cells have large ∼1 μm diameter SGs (∼30 fold larger diameter than synaptic vesicles), respond to stimulation at slower rates (seconds versus milliseconds), demonstrate significant constitutive secretion, and in isolated acini, undergo sequential compound SG–SG exocytosis at the apical membrane. Exocytosis proceeds as an initial rapid phase that peaks and declines over 3 min followed by a prolonged phase that decays to near basal levels over 20–30 min. Studies indicate the early phase is triggered by Ca²⁺ and involves the SG proteins VAMP2 (vesicle associated membrane protein2), Ca²⁺-sensing protein synatotagmin 1 (syt1) and the accessory protein complexin 2. The molecular details for regulation of VAMP8-mediated SG exocytosis and the prolonged phase of secretion are still emerging. Here we review the known regulatory molecules that impact the sequential exocytic process of SG tethering, docking, priming and fusion in acinar cells.     

Restitution of granule stores in the rabbit parotid gland after isoprenaline-induced secretion

Summary A detailed stereological analysis has been made of the organelle content of rabbit acinar cells during the restoration of granule stores following extensive degranulation with isoprenaline (IPR). Rabbits were sacrificed 2, 4, 8, 12 and 16 h after IPR administration and the volumes and proportions of intracellular organelles were compared with those of untreated glands.At 2 h only 5% of cell volume was occupied by secretion granules, but there was already evidence of nascent granule formation. The volume per cell of secretion granules increased in sigmoid fashion and by 16 h amounted to 350 m³/cell, 36% of cell volume, which are values similar to those of the control replete glands. IPR treatment caused some initial swelling of the cells, and there were transient increases in the volumes of several compartments. However, the volume of mitochondrial and lysosomal compartments had returned to control levels by 4 h and that of the nucleus by 8 h. The greatest increase was in the volume of the rough endoplasmic reticulum which had increased by nearly 70% by 2 h and remained enlarged throughout the period of restitution. However, neither the volume nor the proportion of the smooth membraned compartment varied throughout the period of analysis.The results are analysed in the light of the overall response of the cells to IPR and the interaction of the organelles during the synthetic phase of the secretory cycle. They are presented as a basis for ensuing studies of the granule populations and the membrane composition of the cells during the restoration of granule stores.     

Immunolocalization of RhoA and RhoB GTPases in pleomorphic adenoma of the parotid

The pleomorphic adenoma of the parotid (PA) is characterized by the high tissues diversity. Rho GTPases participate in signal transduction pathways that regulate several biological processes, including cell differentiation. A quantitative analysis of RhoA and RhoB GTPases immunoexpression was performed in healthy parotids and in 23 PA cases, predominantly epithelial (PE) or mesenchymal (PM), followed by Student's t test. In PE cases, RhoA immunoexpression was higher in sheets and trabeculae (p < 0.05), whereas RhoB only in sheets (p < 0.05). In normal parotids, RhoA and RhoB were not detected in acinar cells. Ducts have expressed RhoA and RhoB in normal parotids and PA. RhoB was detected in myxoid and chondromyxoid cells. Normal parotids do not express RhoA and RhoB proteins in acinar cells, indicating a lack of function in secretory cells. Despite RhoA and RhoB GTPases are different in their biological roles, no significant difference in immunoexpression of the RhoA and RhoB GTPases in epithelial and mesenchymal structures of PA.     

A primary culture of parotid acinar cells retaining capacity for agonists-induced amylase secretion and generation of new secretory granules

Exocrine acinar cells, like parotid cells, have difficulty in maintaining their functions in cell lines or in primary cultures. For this reason, molecular studies on exocrine cell functions are unsatisfactory. To examine the mechanisms whereby the functions of parotid acinar cells are maintained, we attempted to establish a system for primary culture and transfection of exogenous genes. Acinar cells were dispersed from rat parotid glands by digestion with enzymes and were cultured in a medium containing rat serum. Most of the cultured cells had secretory granules that contained amylase, suggesting that they were derived from acinar cells, although they spread on the dish surface and formed filopodia. The cultured cells retained both granules and the ability to release amylase in response to -adrenergic and cholinergic agonists, even 48 h after dispersion. However, the total amount of amylase in the cells decreased rapidly from 24 to 48 h after dispersion. These results suggested that amylase synthesis was more damaged than the machinery for exocytosis during culture in vitro. VAMP2 gene fused with enhanced green fluorescence protein was transfected into the dispersed acinar cells, and VAMP2 protein was expressed and localized to amylase-containing granules, as normally seen for endogenous VAMP2 protein. This indicated that new granules were generated, and that protein sorting was functional. The cells cultured by this method maintained their functions for at least 48 h. They can be used for examining the effects of exogenous genes on parotid acinar cell functions, such as regulated exocytosis and the maturation of secretory granules.This work was supported in part by Grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (11771148, 13771104, 16390534, 16591868), by Nihon University Multidisciplinary Research Grant for 2001 and 2002, by a Suzuki Memorial Grant of Nihon University School of Dentistry at Matsudo (General Individual Research Grant for 2000 and 2002 and Joint Research Grant for 2003), and by a Grant-in-Aid for a 2001 Multidisciplinary Research Project from MEXT.     

Regulation of calcium handling by rat parotid acinar cells

Summary Salivary gland fluid secretion following neurotransmitter stimulation is Ca²⁺-dependent. We have studied the control of cellular Ca²⁺ following secretory stimuli in rat parotid gland acinar cells. After muscarinic-cholinergic receptor activation, cytosolic Ca²⁺ is elevated 4–5 fold, due to both intracellular Ca²⁺ pool mobilization and extracellular Ca²⁺ entry. Fluid movement ensues due to the Ca²⁺-activated enhancement of membrane permeability to K⁺ and Cl^–. Basal cytosolic Ca²⁺ levels are tightly controlled at 150–200 nM through the action of high affinity and high capacity ATP-dependent Ca²⁺ transporters in the basolateral and endoplasmic reticulum membranes. Activity of these Ca²⁺ transporters can be modulated to facilitate rapid responsiveness and a sustained fluid secretory response necessary for alimentary function.     

Transferrin secretory pathways in rat parotid acinar cells

Transferrin is the major iron transporter in blood plasma, and is also found, at lower concentrations, in saliva. We studied the synthesis and secretion of transferrin in rat parotid acinar cells in order to elucidate its secretory pathways. Two sources were identified for transferrin in parotid acinar cells: synthesis by the cells (endogenous), and absorption from blood plasma (exogenous). Transferrin from both sources is secreted from the apical side of parotid acinar cells. Endogenous transferrin is transported to secretory granules. It is secreted from mature secretory granules upon stimulation with a β-adrenergic reagent and from smaller vesicles in the absence of stimulation. Exogenous transferrin is internalized from the basolateral side of parotid acinar cells, transported to the apical side by transcytosis, and secreted from the apical side. Secretory processes for exogenous transferrin include transport systems involving microfilaments and microtubules.     

Suppression if milk secretion (exocytosis) by concanavalin A in vitro

The observation that concanavalian A can inhibit milk secretion was evaluated in an in vitro system employing minced mammary gland or isolated alveoli from lactating rats. Release of milk constituents (casein, lactose and fat globules) into the medium in the presence and absence of concanavalin A was measured during 1 or 2 h incubations. The effect of concanavalin A on glucose uptake and CO₂ production of the minced tissued was also studied. Concanavalin A suppressed release of milk components at a concentration as low as 80 μg/ml of medium. Respiration of minced mammary tissue in the presence of concanavalin A (100 μg/ml of medium) was essentially the same as that of the control. The data are evidence that concanavalin A acts directly on the mammary cell in suppressing milk secretion and that the effect is not due to cytotoxicity.     

Protease-activated receptor-2 (PAR-2) in the pancreas and parotid gland: Immunolocalization and involvement of nitric oxide in the evoked amylase secretion

Protease-activated receptor-2, a G protein-coupled receptor activated by serine proteases such as trypsin, tryptase and coagulation factors VIIa and Xa, modulates pancreatic and salivary exocrine secretion. In the present study, we examined the distribution of PAR-2 in the pancreas and parotid gland, and characterized the PAR-2-mediated secretion of amylase by these tissues in vivo. Immunohistochemical analyses using the polyclonal antibody against rat PAR-2 clearly showed abundant expression of PAR-2 in rat pancreatic and parotid acini. The PAR-2 agonist SLIGRL-NH2, administered intraperitoneally (i.p.) at 1-10 micromol/kg and 1.5-15 micromol/kg, in combination with amastatin, an aminopeptidase inhibitor, facilitated in vivo secretion of pancreatic and salivary amylase in a dose-dependent manner, respectively, in the mouse. The PAR-2-mediated secretion of pancreatic amylase was abolished by pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor. The secretion of salivary amylase in response to the PAR-2 agonist at a large dose, 15 micromol/kg, but not at a smaller dose, 5 micromol/kg, was partially reduced by L-NAME. Pretreatment with capsaicin for ablation of the sensory neurons did not modify the PAR-2-mediated secretion of pancreatic and salivary amylase in the mouse. In conclusion, our study demonstrates expression of PAR-2 in rat pancreatic acini as well as parotid acini and indicates that nitric oxide participates in the PAR-2-mediated in vivo secretion of pancreatic amylase, and, to a certain extent, of salivary amylase, although capsaicin-sensitive sensory neurons, known to be activated by PAR-2, are not involved in the evoked pancreatic or salivary amylase secretion.     

Crosstalk between purinergic receptors and canonical signaling pathways in the mouse salivary gland

Isolated clusters of mouse parotid acinar cells in combination with live cell imaging were used to explore the crosstalk in molecular signaling between purinergic, cholinergic and adrenergic pathways that integrate to control fluid and protein secretion. This crosstalk was manifested by (1) β-adrenergic receptor activation and amplification of P2X4R evoked Ca²⁺ signals, (2) β-adrenergic-induced amplification of P2X7R-evoked Ca²⁺ signals and (3) muscarinic receptor induced activation of P2X7Rs via exocytotic activity. The findings from our study reveal that purinoceptor-mediated Ca²⁺ signaling is modulated by crosstalk with canonical signaling pathways in parotid acinar cells. Integration of these signals are likely important for dynamic control of saliva secretion to match physiological demand in the parotid gland.