Discharge and restitution of secretory material in the rat parotid gland in response to isoproterenol

Summary The sequence of morphological events occurring during discharge and restitution of secretory material in the rat parotid in response to isoproterenol administration has been studied using the electron microscope. With the dose used, discharge of secretory granules began within 5 min following injection and was complete by 40 mim. Intracellular accumulation of normal-appearing secretory material became evident at 6 hours, and restitution of resting quantities of secretory material was achieved between 12 and 18 hours after injection. Cellular events occurring during secretory discharge and restitution are discussed.This project was supported by Training Grant No. 5-Ti-GH-326 and by Predoctoral Research Grant No. 1-F1-GM-32, 528-01, National Institutes of Health. I wish to express my gratitude to Dr. Henry S. di Stefano under whose directorship this project was carried out.     

Effect of postganglionic sympathectomy on the ultrastructure of the rat parotid gland

Summary Following two weeks of superior cervical ganglionectomy, the parotid glands of adult rats were removed and studied by electron microscopy. Sympathectomy induced striking alterations of acini, resulting in a heterogeneous population of acinar cells, but it had no obvious effect on the duct system. Most of the altered cells could be classified on a cytological basis as dark cells or light cells. Dark cells predominated and contained more secretory granules, less granular endoplasmic reticulum, fewer Golgi membranes, and smaller lumina and intercellular canaliculi than normal acinar cells. The synthesis and extrusion of secretory products appeared to be minimal in these cells. Light cells possessed ultrastructural features, such as dilated cisternae of granular endoplasmic reticulum and prominent Golgi membranes, which were opposite to those of dark cells and indicative of a high degree of secretory activity.The heterogeneous population of cells following sympathectomy indicates that the sympathetic nervous system may play an important role in regulating the secretory synchrony of acinar cells.Supported by U.S.P.H.S. Grant DE 02110.     

Nerve-induced secretion of parotid acinar granules in cats

Summary Electron microscopy of cat parotid glands revealed great heterogeneity in the secretory granules of normal unstimulated acinar cells. Electrical stimulation of the parasympathetic nerve to the gland evoked a copious flow of parotid saliva which was accompanied by an extensive depletion of the secretory granules from the acinar cells. Exocytosis was captured as it was occurring by means of perfusion-fixation, and showed that the events occur in a conventional manner. Stimulation of the sympathetic nerve caused only a very small flow of saliva, and no acinar degranulation was detected. It can be concluded that the parasympathetic secretomotor axons provide the main drive for parotid acinar degranulation in the cat. This contrasts with the rat in which sympathetic impulses provide the main stimulus for parotid acinar degranulation. These dissimilarities serve to emphasise how extensively species differences may influence autonomic responses in salivary glands.     

Changes in the size and number of secretion granules in the rat exocrine pancreas induced by feeding or stimulation in vitro

Summary The size, number and volume per cell of secretion granules in rat exocrine pancreas have been measured using stereological techniques. The changes which occur as a result of feeding starved animals (90 min) or stimulating lobular fragments in vitro with carbachol are documented. In fasted animals mean acinar cell volume was estimated as 1670 m³ and the cells contained an average of around 450 secretion granules with a corrected mean diameter of 0.70 m. They occupied around 7% of cell volume. After feeding mean cell volume was about 1300 m³ and the cells contained an average of about 190 granules per cell with a mean diameter of 0.58 m. They occupied 3% of cell volume. A shift in the size frequency distribution of granule diameters occurred as a result of feeding. In vitro experiments in which lobules were induced to secrete with carbachol (10M, 3 h, 37° C) had a similar effect. Mean cell volume was reduced from around 1760 m³ to 1360 m³, mean granule number from around 420 per cell to 180 per cell and the volume density of granules was reduced from about 8% to 3% of cell volume. There was no significant change in mean granule diameter or shift in the size-frequency distribution of granule diameters. Incubation of tissues with cycloheximide (1 mM, 3 h, 37° C) did not prevent secretion by carbachol but it prevented replacement of granules. As a consequence, depletion by carbachol was greater in the presence of cycloheximide, the granules being reduced to around 110 per cell and to only 2.5% of cell volume. We conclude that feeding causes a preferential loss of larger granules and that during secretion replacement of granules occurs. Some of these granules are smaller than those evident in the glands of starved animals.     

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.     

The ultrastructure of the sheep parotid gland

Summary The sheep parotid is a compound tubular gland; its ultrastructure reflects the function of this gland to secrete large amounts of fluid with very little protein. The cells of the secretory tubules possess extensively folded lateral plasma membranes and a fairly large number of mitochondria. Rapid equilibration of water across the epithelium is assured by the close proximity over large areas of intercellular spaces and the wide secretion canaliculi. Numerous long microvilli extend into the latter. Although secretion granules may be quite numerous, there is evidence that many of these granules are not discharged but undergo degradation by lysosomal enzymes. The intercalated ducts are often dilated but excessive distension is probably prevented by bundles of microfilaments in their epithelial cells.     

Functional mediation of compensatory enlargement of the parotid gland

Summary The role of functional activity in mediating compensatory enlargement of the parotid gland after removal of the other major salivary glands was investigated. Increased levels of activity were achieved by feeding rats a bulk diet. Conversely, a liquid diet was used to reduce the functional demands on the parotid. It was found that the liquid diet completely prevented the compensatory response from occurring. Bulk diet, on the other hand, caused an even greater compensatory response than did the standard chow diet. Compensatory enlargement of the parotid, therefore, depends on its functional activity and not on other, e.g., humoral factors dissociated from function. The character of the cellular response in compensatory enlargement was also examined. The chow diet caused compensatory enlargement by an increase in cell size with little, if any, increase in cell number.Supported in part by the Veterans Administration and Grant DE 02110 of the U.S. Public Health Service     

Characterization of cysteine string protein in rat parotid acinar cells

Cysteine string proteins (CSPs) are secretory vesicle chaperone proteins that contain: (i) a heavily palmitoylated cysteine string (comprised of 14 cysteine residues, responsible for the localization of CSP to secretory vesicle membranes), (ii) an N-terminal J-domain (DnaJ domain of Hsc70, 70 kDa heat-shock cognate protein family of co-chaperones), and (iii) a linker domain (important in mediating CSP effects on secretion). In this study, we investigated the localization of CSP1 in rat parotid acinar cells and evaluated the role of CSP1 in parotid secretion. RT-PCR and western blotting revealed that CSP1 was expressed and associated with Hsc70 in rat parotid acinar cells. Further, CSP1 associated with syntaxin 4, but not with syntaxin 3, on the apical plasma membrane. Introduction of anti-CSP1 antibody into SLO-permeabilized acinar cells enhanced isoproterenol (IPR)-induced amylase release. Introduction of GST-CSP1_1–112, containing both the J-domain and the adjacent linker region, enhanced IPR-induced amylase release, whereas neither GST-CSP1_1–82, containing the J-domain only, nor GST-CSP1_83–112, containing the linker region only, did produce detectable enhancement. These results indicated that both the J-domain and the linker domain of CSP1 are necessary to function an important role in acinar cell exocytosis.     

Neural regulation of compensatory enlargement of the parotid gland of the rat

Summary The role of the innervation in mediating compensatory enlargement of the parotid gland of the rat after partial desalivation was examined. The results of denervation experiments show that full compensatory growth requires both parasympathetic and sympathetic innervation. The presence of the parasympathetic innervation alone results in an increase in the number of cells, but not the size of the cells. The sympathetic innervation alone does not mediate either response. We, therefore, conclude that the two types of innervation have a synergistic action on the parotid to produce the maximal compensatory response, which includes an increase in both number and size of acinar cells.Supported in part by the Veterans Administration and Grant DE 02110 of the U.S. Public Health Service     

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.     

Amylase secretion by rabbit parotid gland role of cyclic AMP and cyclic GMP

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10⁻⁴ M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by α-adrenergic blockade with phenoxybenzamine. Epinephrine (4 · 10⁻⁵ M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the β-blocking agent, propranolol. Pure α-adrenergic stimulation with methoxamine (4 · 10⁻⁴ M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 · 10^{−6 M}, isoproterenol (a β-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 · 10⁻⁵ M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cylcic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 · 10⁻⁶ M).These data strongly suggest that cholinergic muscarinic agonists and α-adrenergic agonist stimulate amylase output in rabbit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by α-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this issue to the effects of cholinergic stimuli.     

Influence of inter-observer delineation variability on radiomic features of the parotid gland

PurposeThis study aimed to quantify the variability in the values of radiomic features extracted from a right parotid gland (RPG) delineated by a series of independent observers.MethodsThis was a secondary analysis of anonymous data from a delineation workshop. Inter-observer variability of the RPG from 40 participants was quantified using DICE similarity coefficient (DSC) and Hausdorff distance (HD). An additional contour was generated using Varian SmartSegmentation. Radiomic features extracted include four shape features, six histogram features, and 32 texture features. The absolute mean paired percentage difference (PPD) in feature values from the expert and participants were ranked . Feature robustness was classified using pre- determined thresholds.Results63% of participants achieved a DSC > 0.7, the auto- segmentation DSC was 0.76. The average HD for the participants was 16.16 mm ± 0.66 mm, and 15.16 mm for the auto-segmentation. 48% (n = 20) and 33% (n = 14) of features were deemed to be robust with a mean absolute PPD < 5%, for the auto-segmentation and manual delineations respectively; the majority of which were from the grey-run length matrix family. 7% (n = 3) of features from the auto- segmentation and 10% (n = 4) from the manual contours were deemed to be unstable with a mean absolute PPD > 50%. The value of the most robust feature was not related to DSC and HD.ConclusionInter-observer delineation variability affects the value of the radiomic features extracted from the RPG. This study identifies the radiomic features least sensitive to these uncertainties. Further investigation of the clinical relevance of these features in prediction of xerostomia is warranted.     

Cyclical changes in the fine structure of bovine endometrial gland cells

Summary Cyclical changes in bovine endometrial gland cells were investigated in six heifers, three at estrus and three at day twelve of the estrous cycle in the luteal phase. The epithelium is generally low at estrus but high in the luteal phase. There are ciliated and non-ciliated cells. The ciliated cells are fewer and lighter and show inconspicuous cyclical changes.The secretory cells show more prominent changes. At estrus, their free border is flat with short microvilli. The conspicuous rough-surfaced endoplasmic reticulum may synthesize protein for later secretion. The Golgi complex seems inactive. The high number of cytosegresomes and dense bodies might express cell regression caused by endocrine changes.In the luteal phase, the cells are lighter with long microvilli. The Golgi apparatus shows vacuoles and immature secretory droplets. Secretory vacuoles with light contents occur in the apical cytoplasm. Some of them appear to discharge their contents into the lumen. This is interpreted as evidence of merocrine secretion. Accumulations of tubular, smooth-surfaced endoplasmic reticulum, masses of glycogen granules, and several fat droplets are present.Some lymphocytes and degranulated granulocytes are seen near the basement membrane, more frequently at estrus.Financial support for this study was received from Anslaget för främjande av medicinsk forskning vid Veterinärhögskolan.The authors express their gratitude to Prof. A. Bane and Dr. J.-E. E. Ringmar, Department of Obstetrics and Gynecology, for their help with the selection and clinical control of the animals and for keeping them in good condition.Post doctoral fellow, No. 43-KO-52 (1968) from the Educational Ministry of Japan.     

Effect of beta-antagonists on isoprenaline-induced secretion of fluid, amylase and protein by the parotid gland of the red kangaroo, Macropus rufus

Selective and non-selective beta-adrenoceptor antagonists were used to block the increases in fluid, protein and amylase secretion caused by sympathomimetic stimulation of the parotid gland of red kangaroos during intracarotid infusion of isoprenaline. ICI118551 at antagonist/agonist ratios up to 300:1 caused increasing but incomplete blockade of fluid secretion, and protein/amylase release. Atenolol at antagonist/agonist ratios up to 300:1 was only marginally more potent than ICI118551 at blocking the fluid, protein and amylase responses. Propranolol at antagonist/agonist ratios of 30:1 was as effective at blocking fluid and protein secretion as the highest ratios of either atenolol or ICI118551. Simultaneous administration of atenolol (30:1) with ICI118551 (30:1) was not as potent as propranolol (30:1). Thus, the beta-adrenoceptor/s in the acini of the kangaroo parotid gland appear to have antagonist-binding affinities atypical of those found for eutherian tissues. The data are consistent with the gland possessing either a single anomalous beta-adrenoceptor or functional beta(2)-receptors in addition to the beta(1)-receptors which are characteristic of eutherian salivary glands.     

Uncharted cholinergic nerves in the rabbit parotid gland.

Cholinergic nerves are shown to be left in the rabbit parotid gland after avulsion of the auriculo-temporal nerve: a cholinesterase inhibitor injected through the duct caused secretion, thereby revealing leakage of acetylcholine from cholinergic nerve endings, and acetylcholinesterase positive nerves were found histochemically. The incomplete cholinergic denervation offers an explanation to the fact that some choline acetyltransferase activity remains in the 'denervated' glands.     

Amylase secretion by rabbit parotid gland. Role of cyclic AMP and cyclic GMP.

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10(-4) M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by alpha-adrenergic blockade with phenoxybenzamine. Epinephrine (4 - 10(-5) M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the beta-blocking agent, propranolol. Pure alpha-adrenergic stimulation with methoxamine (4 - 10(-4) M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 - 10(-6) M, isoproterenol (a beta-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 - 10(-5) M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cyclic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 - 10(-6) M). These data strongly suggest that cholinergic muscarinic agonists and alpha-adrenergic agonists stimulate amylase output in rabit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by alpha-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this tissue to the effects of cholinergic stimuli.     

Anion secretion by the isolated rabbit pancreas

The isolated rabbit pancreas secretes a fluid containing chloride and bicarbonate in about equal concentrations. Replacement of bicarbonate by acetate, phosphate or isethionate, replacement of Na⁺ by Li⁺ and addition of ouabain to the bathing medium of the pancreas inhibit the secretion of fluid, chloride and bicarbonate in a similar fashion and by maximally 100%. Replacement of chloride by isethionate inhibits fluid secretion by maximally 50%, chloride secretion by 90% and bicarbonate secretion by 20%. It is concluded that fluid secretion is based on a Na⁺-gradient-dependent bicarbonate influx or proton efflux in the ductular cell, and that the secretion of chloride is secondary to that of bicarbonate.     

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.     

Early effects of prolactin on lactating rabbit mammary gland

Summary Effects of prolactin on the secretion of milk proteins have been investigated by incubating mammary tissue fragments from lactating rabbits. Within 15min of adding the hormone to the incubation medium, cell morphology is modified: the relative volume occupied by the Golgi region is greatly increased. When prolactin is added immediately after a pulse labelling of proteins (3 min with ³H-L-leucine), the amount of labelled caseins secreted during one hour is significantly increased. This increase proceeds neither from an acceleration of intracellular transit of caseins (as shown by electron microscopic autoradiography) nor by an enhancement of amino acid uptake (as measured by incorporation of non-metabolizable amino acids) nor by an increase of overall protein synthesis, during the first hour.The action of prolactin on the morphology of such subcellular organelles as the Golgi apparatus and its influence on casein secretion are discussed.We are extremely grateful to Professor Hubert Clauser for his helpful advice. We thank Mrs. Georgette Gianni for her excellent technical assistance and Miss Aline Solari for her contribution to statistical analysis     

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.