Cytochemical detection of polysaccharides on mouse parotid acinar cells

Summary Cytochemical detection of surface polysaccharides on mouse parotid acinar cells was carried out by sequential incubation of fixed slices in periodic acid, thiosemicarbazide and osmium tetroxide. Mice were previously treated with isoproterenol (0.67 moles or 1.5 nmoles per g bd wt), pilocarpine (0.27 moles per g bd wt) or saline.Parotid glands from control mice showed acinar cells with a strong positive reaction at the apical, lateral and basal surfaces. The osmium deposits on the cell surface formed parallel rows of dense granules large enough to permit an easy identification of the polysaccharides.A low dose of isoproterenol and pilocarpine that induced only secretion, produce a slight reduction of the reactivity at the apical cell surface 30 min and 2 h after injection. No other change in the reactivity of the cell surface polysaccharides was found in the times observed.The high dose of isoproterenol that induces secretion and cell proliferation, provoked at 30 min the same lower reactivity at the apical surface as described above. However, at 12 and 20 h after the high dose of isoproternol there is a marked decrease or no reactivity at the apical and lateral cell surfaces. At 36 h, no reactivity was observed at the apical, lateral nor basal surface.The results presented here strongly suggest that polysaccharides are removed from the surface of acinar parotid cells, or that the cell surface is exchanged with inmature secretory vesicles, upon stimulation to secretion and cell proliferation. These polysaccharides are not affected or lost, with stimuli that induce only secretion.This work was supported by Research Grants #4147-R from the Servicio de Desarrollo Científico y Creación Artística, Universidad de Chile and #14-77 from the Programa Regional de Entrenamiento para Países del Area Andina RLA/047 (PNUD/UNESCO)This paper is dedicated to Professor Dr. Danko Brncic on the occasion of his 30 years of academic work.     

Cell-specific behavior of P2X7 receptors in mouse parotid acinar and duct cells

P2X7 receptors (P2X7Rs) affect many epithelial cell functions including transcellular ion transport, secretion, and cell death. Here we used parotid acinar and duct cells to reveal the unique cell-specific assembly and gating of the P2X7R channels. Immunolocalization indicated expression of P2X7Rs in the luminal membrane of both cell types. Stimulation with 5 mm ATP raised [Ca2+]i levels in a cell-specific manner and activated multiple currents. The current mediated by P2X7R was isolated by infusing the cells with high [EGTA]. The initial activation of acinar cell P2X7Rs by ATP was slow requiring approximately 2.5 min. Subsequent removal and addition of ATP, however, resulted in rapid inhibition and activation (gating) of the P2X7Rs. By contrast, P2X7Rs in duct cells displayed only rapid gating by ATP. Activation of P2X7Rs in both cell types was verified by (a) low Km for ATP, (b) sensitivity to external divalent ions, (c) lack of desensitization/inactivation, (d) permeability to Na+, and (e) inhibition by Brilliant Blue G, Cu2+, and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium. The slow P2X7R activation in acinar cells was not affected by manipulation of exo-/endocytosis. Rather, disassembly or solidification of the actin cytoskeleton prior to incubation with ATP prevented channel assembly. Remarkably, after completion of the slow activation, manipulation of the actin cytoskeleton no longer affected gating by ATP. Accordingly, manipulation of the actin cytoskeleton had no effect on P2X7R gating by ATP in duct cells. We concluded that P2X7Rs are not active in resting acinar cells. On exposure to ATP, P2X7Rs are assembled into functional channels with the aid of the actin cytoskeleton. Once assembled, P2X7Rs are subject to rapid gating by ATP. Duct cell P2X7Rs are preassembled and therefore continually subject to rapid gating by ATP. This cell-specific behavior may reflect the specific function of P2X7Rs in the two cell types.     

Cytochemical localization of complex carbohydrates in the thyroid gland of normal and TSH-treated mice

Summary The silver methenamine method for the ultrastructural localization of carbohydrates and glycoproteins was applied to the thyroid glands of normal and TSH-treated mice. The majority of the cisternae of the rough endoplasmic reticulum showed a weak, but apparently positive reaction. These findings support the opinion that glycosylation of thyroglobulin occurs initially in the rough endoplasmic reticulum. By this method the Golgi apparatus was observed to display a staining gradient. The intermediate to inner saccules were intensely stained, whereas the outer saccules were not so heavily stained. This phenomenon indicates that the Golgi apparatus has a functional polarity for the addition of carbohydrates to thyroglobulin and other proteins. In the inner and/or the peripheral regions of the Golgi apparatus and in the apical cytoplasm, a large number of globules of various sizes, considered to be colloid droplets, lysosomes and apical secreting vesicles, showed a positive reaction. The luminal colloid was also positive with silver methenamine staining, with almost the same intensity as the globules and vesicles.This study was supported by a grant from the Japan Ministry of Education     

Cytochemical localization of acid phosphatase and trimetaphosphatase activities in exocrine acinar cells

Acid phosphatase activity, a lysosomal marker, is commonly demonstrated using the Gomori technique with cytidine 5'-monophosphate or beta-glycerophosphate as substrate. Using this lead capture method on mouse and rat exorbital lacrimal, parotid, and pancreatic acinar cells, reaction product was localized in GERL, forming secretory granules, and secondary lysosomes. However, a different cytochemical localization was observed for inorganic trimetaphosphatase, another lysosomal enzyme. When the technique for trimetaphosphatase activity, a metal chelation method, was applied to exocrine acinar cells, reaction produce was conspicuously absent from GERL and forming secretory granules, but was present in secondary lysosomes, occasionally in Golgi saccules, and in previously unreported basal elongated lysosomes. The differences in the localization of the two enzymatic activities emphasizes the importance of employing more than one substrate where possible, and raises questions concerning the mechanism of delivery of acid hydrolases to secondary lysosomes.     

Vitamin D receptors in isolated rat parotid gland acinar cells

Rat parotid gland was examined for the presence of 1α,25-dihydroxycholecalciferol receptors using sucrose density gradient ultracentrifugation techniques. [³H]DHCC bound specifically and with high affinity to a 3.2 S protein present in nuclear and cytosolic fractions of isolated parotid acinar cells. Values for the equilibrium dissociation constant and for the receptor concentration were determined to be approx. 0.1 nM, and 12 fmol/mg protein, respectively. In competitive inhibition experiments, the 3.2 S protein displayed 100-fold lower affinity for 25-hydroxycholecalciferol than for DHCC, and did not bind estradiol or methylprednisolone. These results suggest that rat parotid gland acinar cells contain classical DHCC receptors. A similar approach failed to provide evidence of DHCC receptors in isolated pancreas acinar cells, lacrimal gland or submandibular gland. It has been previously reported that vitamin D is essential for normal exocrine secretion from the rat parotid gland (Tenenhouse, A. and Afari, G. (1978) Biochim. Biophys. Acta 538, 631–634). The present findings suggest that this effect is the result of a direct action of DHCC on the parotid gland acinar cell. The absence of DHCC receptors in other exocrine cells suggests that tissue sensitivity to DHCC is not a general property of exocrine systems.     

Cytochemical detection of endogenous peroxidase in the acinar cells of the hamster submandibular gland

The presence of endogenous peroxidase activity in the hamster submandibular gland was investigated cytochemically by light and electron microscopy using diaminobenzidine methods. After fixation of tissue with 2% paraformaldehyde--2.5% glutaraldehyde and incubation in a DAB reaction medium containing 0.01% H₂O₂, the peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, secretory granules and the Golgi apparatus in both the acinar and granular duct cells of the submandibular gland. This is in contrast to earlier investigators who failed to detect peroxidase activity in acinar cells of the hamster submandibular gland and reported that peroxidase is localized only in the granular duct cells. The discrepancy may be caused by differences in experimental procedures. It is suggested that fixation of tissue with a high concentration of glutaral dehyde and incubation in a DAB reaction medium containing a high concentration of H₂O₂ inhibits the peroxidase activity of acinar cells in the hamster submandibular gland     

Cytochemical detection of endogenous peroxidase in the acinar cells of the hamster submandibular gland

The presence of endogenous peroxidase activity in the hamster submandibular gland was investigated cytochemically by light and electron microscopy using diaminobenzidine methods. After fixation of tissue with 2% paraformaldehyde--2.5% glutaraldehyde and incubation in a DAB reaction medium containing 0.01% H₂O₂, the peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, secretory granules and the Golgi apparatus in both the acinar and granular duct cells of the submandibular gland. This is in contrast to earlier investigators who failed to detect peroxidase activity in acinar cells of the hamster submandibular gland and reported that peroxidase is localized only in the granular duct cells. The discrepancy may be caused by differences in experimental procedures. It is suggested that fixation of tissue with a high concentration of glutaral dehyde and incubation in a DAB reaction medium containing a high concentration of H₂O₂ inhibits the peroxidase activity of acinar cells in the hamster submandibular gland This revised version was published online in November 2006 with corrections to the Cover Date.     

Cytochemical demonstration of actin filaments in myoepithelial cells of the human parotid gland

Ultrastructurally, myoepithelial cells were shown to contain numerous fine filaments in their cytoplasm and resembled smooth muscle cells. The myoepithelial cell of the salivary gland has been considered to play an important role in the secretion of saliva. The present study showed that all the thin filaments (actin filaments) in the myoepithelial cell of the human parotid gland bound heavy meromyosin (HMM) and formed characteristic arrowhead structures. These filaments ran in two opposite directions with the poles at different ends. On the other hand, there was no binding of HMM with thicker filaments (10-nm filaments), plasma membrane, nuclear membrane, collagen fibrils, basement membrane or other cytoplasmic organelles. The present results strongly suggest that myoepithelial cells possess a contractile function parallel to the long axis of the cell for supporting the secretion of saliva in the parotid gland.     

Expression and localization of rab escort protein isoforms in parotid acinar cells from rat

Rab proteins are geranylgeranylated on their carboxyl terminal cysteine motifs by geranylgeranyltransferase II (GGTase). Rab escort protein (REP) is required to present Rab proteins to GGTase. REP may remain bound to newly isoprenylated Rab proteins and present them to their target membrane. Other studies have shown that Rab proteins cycle between the membrane and cytosolic compartments and that cytosolic Rab proteins are complexed with rab-GDI. In the present study, we examined the expression and localization of REP isoforms in parotid acinar cells. Although both REP isoforms, REP-1 and REP-2, were detected in parotid cytosol, REP-2 was the predominant isoform. Subcellular fractionation revealed that approximately 42% of cellular REP-2 is membrane-associated. REP-2 was partially removed from parotid membranes with 1 M NaCl or Na(2)CO(3), indicating that REP-2 is a peripheral membrane protein. Membrane-associated REP-2 did not colocalize with Rab3D on secretory granule membranes. However, density gradient centrifugation revealed that membrane-associated REP-2 and Rab3D colocalize on low- and high-density membrane fractions in parotid acinar cells. Isoproterenol, an agent which induces amylase release from parotid glands, caused a shift in both REP-2 and Rab3D to less dense membrane fractions. When acinar cell cytosol was fractionated by gel filtration chromatography, Rab3D eluted exclusively with REP, not rab-GDI. In contrast, Rab1B and Rab5 eluted with both REP and Rab-GDI. Colocalization of Rab3D and REP-2 on acinar cell membranes suggests that REP-2 plays a role in delivering Rab3D to parotid membranes and may regulate guanine nucleotide binding to membrane-associated Rab3D. In addition, unlike other Rab proteins, cytosolic Rab3D appears to associate exclusively with REP, not rab-GDI in parotid acinar cells.     

Intracellular pH regulation in the mouse lacrimal gland acinar cells

Summary Intracellular pH (pH _i ) of the acinar cells of the isolated, superfused mouse lacrimal gland has been measured using pH-sensitive microelectrodes. Under nonstimulated condition pH _i was 7.25, which was about 0.5 unit higher than the equilibrium pH. Alterations of the external pH by ±0.4 unit shifted pH _i only by ±0.08 unit. The intracellular buffering value determined by applications of 25mm NH ₄ ⁺ and bicarbonate buffer solution gassed with 5% CO₂/95% O₂ was 26 and 46mm/pH, respectively Stimulation with 1 m acetylcholine (ACh) caused a transient, small decrease and then a sustained increase in pH _i . In the presence of amiloride (0.1mm) or the absence of Na⁺, application of ACh caused a significant decrease in pH _i and removal of amiloride or replacement with Na⁺-containing saline, respectively, rapidly increased the pH _i . Pretreatment with DIDS (0.2mm) did not change the pH _i of the nonstimulated conditions; however, it significantly enhanced the increase in pH _i induced by ACh. The present results showed that (i) there is an active acid extrusion mechanism that is stimulated by ACh; (ii) stimulation with ACh enhances the rate of acid production in the acinar cells; and (iii) the acid extrusion mechanism is inhibited by amiloride addition to and Na⁺ removal from the bath solution. We suggest that both Na⁺/H⁺ and HCO ₃ ^– /Cl^– exchange transport mechanisms are taking roles in the intracellular pH regulation in the lacrimal gland acinar cells.     

Lectin-binding pattern in parotid acinar cells

Summary The localization of complex carbohydrates in the Golgi apparatus, secretory granules and plasmalemma of mouse parotid acinar cells was studied using the fracture-labelling method. The hexose residues of glycoconjugates were identified using ferritin conjugated with Wheat Germ Agglutinin (WGA-), Ricinnus Communis Agglutinin II (RCA-II-), Phaseolus Vulgaris Agglutinin (PHA-) and Limulus Polyphemus Agglutinin (LPA-). We found that the tracture-labelling method allows not only the labelling of membrane faces but also analysis of the compartment's content that is exposed during the fracturing of the tissue. Our results revealed differences in the hexose residues located in the Golgi apparatus, secretory granules and the apical and lateral plasmalemma. Numerous binding sites for WGA-, PHA-and RCA-II-ferritin were demonstrable in the Golgi apparatus. In secretory granules, the WGA-and RCA-II-ferritin binding sites were most numerous, while LPA-ferritin binding sites were very rate. The density of the binding sites for PHA-ferritin showed considerable variation in secretory granules. The apical plasmalemma exhibited a high density of binding sites for all of the lectins used. In the lateral plasmalemma, LPA-ferritin was not bound, and there were fewer binding sites for WGA-, RCA-H-and PHA-ferritin.     

Apical Ca2+-activated potassium channels in mouse parotid acinar cells

Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca(2+)] was used to investigate if Ca(2+)-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca(2+)-buffering conditions that produced brief, localized increases in [Ca(2+)] after focal laser photolysis of caged Ca(2+). Conditions were used to isolate K(+) and Cl(-) conductances. Photolysis at the apical PM resulted in a robust increase in K(+) and Cl(-) currents. A localized reduction in [Ca(2+)] at the apical PM after photolysis of Diazo-2, a caged Ca(2+) chelator, resulted in a decrease in both K(+) and Cl(-) currents. The K(+) currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance "maxi-K" (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34-sensitive K(+) currents were also observed in BK-null parotid acini. In contrast, when the [Ca(2+)] was increased at the basal or lateral PM, no increase in either K(+) or Cl(-) currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.     

Ultrastructural morphometric study on developing acinar cells of the rat pancreas and parotid gland

The fraction of cellular volume occupied by the cytoplasm has been assessed by point-counting volumetry in 0.5 micrometer araldite sections. Measurement of the transection radius of the nuclei of acinar cells allowed the assessment of the mean nuclear volume according to the method of Bach. With these data, the cytoplasm volume was calculated in cubic micrometers. Ultrastructural morphometric data were obtained by means of superimposing on the electron photomicrographies (x 21,000 or x 28,500) a test system of 84 evenly spaced segments according to Weibel et al. The analysis of the values obtained for the pancreas and parotid gland allowed for the supposition that the process of maturation in the rat, either for the pancreas or the parotid gland, occurs simultaneously with the mitotic process, this last being held as the predominating one in the first 3--4 postnatal weeks. The cytodifferentiation process becomes more marked in the last phases studied and on the 40th day it already shows parameters held as similar to those of the adult animal.     

Endogenous retinoic acid activity in principal cells and intercalated cells of mouse collecting duct system

Background

Retinoic acid is the bioactive derivative of vitamin A, which plays an indispensible role in kidney development by activating retinoic acid receptors. Although the location, concentration and roles of endogenous retinoic acid in post-natal kidneys are poorly defined, there is accumulating evidence linking post-natal vitamin A deficiency to impaired renal concentrating and acidifying capacity associated with increased susceptibility to urolithiasis, renal inflammation and scarring. The aim of this study is to examine the presence and the detailed localization of endogenous retinoic acid activity in neonatal, young and adult mouse kidneys, to establish a fundamental ground for further research into potential target genes, as well as physiological and pathophysiological roles of endogenous retinoic acid in the post-natal kidneys.

Methodology/Principal Findings

RARE-hsp68-lacZ transgenic mice were employed as a reporter for endogenous retinoic acid activity that was determined by X-gal assay and immunostaining of the reporter gene product, β-galactosidase. Double immunostaining was performed for β-galactosidase and markers of kidney tubules to localize retinoic acid activity. Distinct pattern of retinoic acid activity was observed in kidneys, which is higher in neonatal and 1- to 3-week-old mice than that in 5- and 8-week-old mice. The activity was present specifically in the principal cells and the intercalated cells of the collecting duct system in all age groups, but was absent from the glomeruli, proximal tubules, thin limbs of Henle''s loop and distal tubules.

Conclusions/Significance

Endogenous retinoic acid activity exists in principal cells and intercalated cells of the mouse collecting duct system after birth and persists into adulthood. This observation provides novel insights into potential roles for endogenous retinoic acid beyond nephrogenesis and warrants further studies to investigate target genes and functions of endogenous retinoic acid in the kidney after birth, particularly in the collecting duct system.     

Adrenergic and cholinergic mediated glucose oxidation by rat parotid gland acinar cells during aging

A₂-phospholipase from Crotalus Adamanteus venom has been assayed using the fluorescent probe 2-all trans-parinoyllecithin. The hydrolysis of the lecithin in an “albumin-rich” incubation medium is paralleled with a fluorescence hyperpolarization. The mechanism of the hyperpolarization is discussed. The application of the method to assay phospholipases, allows detection of an activity under 1 nanomole/min and continuous monitoring of the hydrolysis.