Tight junctional permeability of the resting and carbachol stimulated exocrine rabbit pancreas

Summary The permeability of the pancreatic epithelium to horseradish peroxidase is investigated in the resting and carbachol stimulated rabbit pancreas. Horse radish peroxidase administered to the bathing medium of the isolated rabbit pancreas appears in the secreted fluid of the pancreas in a relatively low concentration. Carbachol stimulates both protein secretion and the passage of horse radish peroxidase into the secretory fluid. Histochemical assessment shows that horseradish peroxidase enters the interstitial spaces of the pancreatic tissue and is present along basal and lateral plasma membranes of acinar and ductular cells. In the absence of carbachol, horseradish peroxidase is seen more frequently in the tight junctions of ductular cells than in those of acinar cells. However, in the carbachol stimulated gland horseradish peroxidase is observed in the junctions between adjacent acinar cells more frequently than in the unstimulated gland. Freeze-fracture of acinar cells shows that the number of tight junctional strands and the tight junction depth are slightly decreased upon carbachol stimulation. The findings suggest that cholinergic stimulation of the exocrine pancreas increases the permeability of the acinar cell junctions to moderately large molecules such as horseradish peroxidase. This may result in an increase of the concentration of the molecule in the secreted fluid.     

Tight junctional permeability of the resting and carbachol stimulated exocrine rabbit pancreas

The permeability of the pancreatic epithelium to horseradish peroxidase is investigated in the resting and carbachol stimulated rabbit pancreas. Horse radish peroxidase administered to the bathing medium of the isolated rabbit pancreas appears in the secreted fluid of the pancreas in a relatively low concentration. Carbachol stimulates both protein secretion and the passage of horse radish peroxidase into the secretory fluid. Histochemical assessment shows that horseradish peroxidase enters the interstitial spaces of the pancreatic tissue and is present along basal and lateral plasma membranes of acinar and ductular cells. In the absence of carbachol, horseradish peroxidase is seen more frequently in the tight junctions of ductular cells than in those of acinar cells. However, in the carbachol stimulated gland horseradish peroxidase is observed in the junctions between adjacent acinar cells more frequently than in the unstimulated gland. Freeze-fracture of acinar cells shows that the number of tight junctional strands and the tight junction depth are slightly decreased upon carbachol stimulation. The findings suggest that cholinergic stimulation of the exocrine pancreas increases the permeability of the acinar cell junctions to moderately large molecules such as horseradish peroxidase. This may result in an increase of the concentration of the molecule in the secreted fluid.     

Uptake and fate of luminally administered horseradish peroxidase in resting and isoproterenol-stimulated rat parotid acinar cells

The formation and fate of apical endocytic vesicles in resting and isoproterenol-stimulated rat parotid acinar cells were studied using luminally administered horseradish peroxidase (HRP) to mark the vesicles. The tracer was taken up from the lumen by endocytosis in small, smooth-surfaces "c"- or ring-shaped vesicles. About 1 h after HRP administration the vesicles could be found adjacent to the Golgi apparatus. At later times HRP reaction product was localized in multivesicular bodies and lysosomes; in isoproterenol-stimulated cells it was also present in autophagic vacuoles. HRP reaction product was never localized in any structure associated with secretory granule formation. These results suggest that the apical endocytic vesicles play a role in membrane recovery, but that they are degraded and not reutilized directly in secretory granule formation. Additionally, it was found that when isoproterenol was injected before HRP administration, the apical junctional complexes became permeable to the tracer, allowing it to gain access to the lateral and basal intercellular spaces. This permeability may provide an additional route whereby substances in the extracellular fluid could reach the saliva.     

Effect of nitrendipine, a calcium antagonist, on cell volume in rat salivary glands after isoproterenol stimulation.

Four days of isoproterenol injections induced a marked enlargement of the rat parotid and submandibular glands reflected in significant increases in the absolute and relative wet and dry weight of the glands. The enlargement in parotid gland was attributable at least in part to cellular hypertrophy inasmuch as the average volume per cell of acinar cells increased. In contrast, the average volume of acinar cells in the submandibular gland was decreased as compared to that of control. It is likely that hyperplasia in both groups accounts in part for the enlargement. The slow calcium channel is unlikely involved in the isoproterenol-induced stimulation of the gland, inasmuch as the calcium channel antagonist did not modify the enlargement of the parotid or submandibular glands.     

Endocytic pathways at the lateral and basal cell surfaces of exocrine acinar cells

In parotid acinar cells, horseradish peroxidase (HRP) administered via the main excretory duct is endocytosed from the apical cell surface in smooth C- or ring-shaped vesicles (Oliver, C. and A. R. Hand. 1979. J. Cell Biol. 76:207). These vesicles ultimately fuse with lysosomes adjacent to the Golgi apparatus. The present investigation extends these findings and examines the uptake and fate of intravenously injected HRP from the lateral and basal cell surfaces of resting and stimulated parotid and pancreatic acinar cells from rats and mice. Isoproterenol and pilocarpine were used to stimulate the parotid gland and the pancreas, respectively. HRP was internalized in smooth and coated vesicles primarily in areas of membrane infoldings. Both the number of coated vesicles and the amount of tracer internalized increased markedly following secretagogue administration. In both resting and stimulated cells, the HRP was rapidly sequestered in a unique system of basally located lysosomes that possess trimetaphosphatase activity, but not acid phosphatase activity. At 1-3 h after HRP administration, reaction product was also found in multivesicular bodies, vesicles, and lysosomes adjacent to the Golgi apparatus. With time, more HRP was localized in Golgi-associated lysosomes. By 6-7 h, tubules in the apical cytoplasm of stimulated cells contained HRP reaction product. When native ferritin was administered retrogradely and HRP injected intravenously, both tracers could be localized in the same lysosome after 4-5 h, indicating that material taken in from all cell surfaces mixes in Golgi-associated lysosomes. The results of this study suggest that two separate and distinct endocytic pathways exist in exocrine acinar cells: one involves membrane retrieval from the apical cell surface; and the other is a stimulation-dependent process at the lateral and basal cell surfaces.     

Early events of secretory granule formation in the rat parotid acinar cell under the influence of isoproterenol. An ultrastructural and lectin cytochemical study

The events involved in the maturation process of acinar secretory granules of rat parotid gland were investigated ultrastructurally and cytochemically by using a battery of four lectins [Triticum vulgaris agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), Glycine max agglutinin (SBA), Arachys hypogaea agglutinin (PNA)]. In order to facilitate the study, parotid glands were chronically stimulated with isoproterenol to induce secretion. Specimens were embedded in the Lowicryl K4M resin. The trans-Golgi network (TGN) derived secretory granules, which we refer to as immature secretory granules, were found to be intermediate structures in the biogenesis process of the secretory granules in the rat parotid acinar cell. These early structures do not seem to be the immediate precursor of the mature secretory granules: in fact, a subsequent interaction process between these early immature granule forms and TGN elements seems to occur, leading, finally, to the mature granules. These findings could explain the origin of the polymorphic subpopulations of the secretory granules in the normal acinar cells of the rat parotid gland. The lectin staining patterns were characteristic of each lectin. Immature and mature secretory granules were labelled with WGA, SBA, PNA, and lightly with UEA-I. Cis and intermediate cisternae of the Golgi apparatus were labelled with WGA, and trans cisternae with WGA and SBA.     

Alpha-lactalbumin acts as a bimodal regulator of rat parotid acinar cell growth

Isoproterenol, a beta-adrenergic receptor agonist, causes hypertrophy and hyperplasia of the rat parotid gland. The stimulation of parotid acinar cells to a growth phase is accompanied by a cell surface localization of the enzyme 4 beta-galactosyltransferase. Alpha-lactalbumin, a specific modifier protein for 4 beta-galactosyltransferase, when given subsequent to the initiation of isoproterenol treatment and the commencement of parotid enlargement, resulted in a termination of gland hypertrophy and DNA synthesis. Gland size did not, however, return to control levels with the continued injection of isoproterenol and alpha- lactalbumin. In contrast, the injection of alpha-lactalbumin in neonatal rats (7-14 days post-partum) stimulated parotid gland hypertrophy and DNA synthesis. This treatment also lead to the precocious expression of the major parotid gland salivary enzyme, amylase.     

Localization of cAMP-dependent protein kinase subunits along the secretory pathway in pancreatic and parotid acinar cells and accumulation of the catalytic subunit in parotid secretory granules following beta-adrenergic stimulation

Catalytic (C) and regulatory (RI and RII) subunits of cAMP-dependent protein kinases were localized by immunoelectron microscopy in cisternae of the rough endoplasmic reticulum (rER) and in the Golgi complex of rat pancreas or parotid cells. Zymogen granules of the exocrine pancreas showed C- and RI-immunoreactivity, secretory granules of parotid acinar cells only RII-immunoreactivity. Injection of rats with isoproterenol (IPR) increased in the parotid gland the number of acinar cells with RII-labeled granules. In addition, it led to the appearance of C-immunoreactivity in the condensing vacuoles and secretory granules with a maximum at 24 h after stimulation. This was confirmed by enzyme-linked immunosorbent assay (ELISA) determinations of C- and RII-subunits in secretory granules isolated from stimulated and control parotid glands. The amount of immunoreactive C-subunits in the secretory granules increased further following repeated injections of the beta-agonist. These findings suggest the existence of secretory forms of cAMP-dependent protein kinase R- and C-subunits and their separate regulation.     

Tight junctions in the rat parotid gland

The aim of this study was to elucidate the distribution and morphological changes of tight junctions during secretion in parotid gland acinar cells. Localization of tight junction-associated polypeptide ZO-1, and of tight junction transmembrane protein Occludin, was examined in rat parotid gland by immunofluorescence and immunogold labelling of ultrathin sections. Adult male Sprague-Dawley rats were intraperitoneally injected with IPR and, after 10 and 30 minutes, parotid glands were extirpated. In control specimens, positive immunoreaction for ZO-1 and Occludin was observed on the adluminal side between adjacent cells in the form of narrow elongated profiles corresponding to intercellular canaliculi. After IPR injection, canaliculi became dilated and fluorescence was no longer seen as a continuous line but appeared as an aggregation of separate bright particles. ZO-1 was more widely distributed and was recognized in other areas of the cytoplasm as well. Concurrently, omega-shaped concavities, marked by actin fluorescence, appeared along the intercellular canaliculi. We concluded that, during exocytosis, the selective permeability barrier to the paracellular pathway, based on tight junctions, becomes more leaky, owing to segregation of Occludin caused by intracellular ZO-1 distributional changes associated with actin filaments.     

Connexins in salivary glands

Connexins (Cxs) make up a family of gap junction structural proteins that form hexameric assemblies in the plasma membranes of adjacent cells that interact to form intercellular channels. It has been demonstrated that many kinds of CXs are differentially expressed in a variety of tissues; however, there have been only a few studies of CX expression in rat salivary glands. The co-localization of CX26 and 32 was examined in the parotid glands. Double immunofluorescence revealed that CX26 and 32 were present in the same gap junction. Double immuno-electron microscopy showed co-localization of both CX26 and 32 on the same gap junctional membranes between acinar cells. These results suggest that CX26 and 32 may participate in regulation of secretory function and permeability of acinar cells in the rat parotid glands.     

Aquaporin-5 (AQP5), a water channel protein, in the rat salivary and lacrimal glands: immunolocalization and effect of secretory stimulation

Aquaporin-5 (AQP5) is a water channel protein and is considered to play an important role in water movement across the plasma membrane. We raised anti-AQP5 antibody and examined the localization of AQP5 protein in rat salivary and lacrimal glands by immunofluorescence microscopy. AQP5 was found in secretory acinar cells of submandibular, parotid, and sublingual glands, where it was restricted to apical membranes including intercellular secretory canaliculi. In the submandibular gland, abundant AQP5 was also found additionally at the apical membrane of intercalated duct cells. Upon stimulation by isoproterenol, apical staining for AQP5 in parotid acinar cells tended to appear as clusters of dots. These results suggest that AQP5 is one of the candidate molecules responsible for the water movement in the salivary glands.     

Regulation of proto-oncogenes in rat parotid acinar cells in vitro after stimulation of beta-adrenergic receptors

Stimulation of beta-adrenoreceptors in rat parotid acinar cells in vitro by the beta-adrenergic agonist isoproterenol induces steady-state levels of c-fos mRNA and c-fos protein in these cells. A dramatic increase in the steady-state levels of c-fos mRNA was observed at 60 min, followed by a decrease at 2 h with a second peak at 4 h. c-fos induction in rat parotid acinar cells in vitro seems to be mediated by cAMP. Increased levels of p53 and c-myc mRNA were detected only at 60 min. c-abl and c-sis were also induced by isoproterenol but in a pattern different from that seen with c-fos. c-abl was the only oncogene in rat parotid gland which showed increased expression after chronic isoproterenol treatment of rats. In rat parotid acinar cells we observed no correlation between DNA synthesis and c-fos induction.     

Mobilization of the hormone-sensitive calcium pool increases hepatocyte tight junctional permeability in the perfused rat liver

Hepatocyte tight junctional permeability has been shown to be regulated by hormones that exert their effects via phospholipase C activation. However, the precise transduction pathway involved in this effect is not known. The present study has employed the selective inhibitor of microsomal Ca2+ sequestration, 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), to examine the effect of the mobilization of the endoplasmic reticular Ca2+ pool on tight junctional permeability in the perfused rat liver. Infusion of tBuBHQ followed by a bolus infusion of horseradish peroxidase (HRP) resulted in a significant increase in the first peak of biliary HRP, a measure of junctional permeability, whereas transcellular (vesicular) transport of HRP was not affected. Therefore, we conclude that the effect of hormones on tight junctional permeability is mediated, at least in part, by the mobilization of intracellular Ca2+.     

Down-regulation of cellular proto-oncogenes during inhibition of rat parotid acinar cell proliferation.

The role of cell surface galactosyltransferase in mediating isoproterenol-induced parotid gland hypertrophy and hyperplasia was examined in rat parotid gland acinar cells. Introduction of the transferase modifier, alpha-lactalbumin, or galactosyltransferase-associated kinase inhibitor trifluoperazine, into beta-agonist-treated rats prevented acinar cell proliferation as determined by [3H]thymidine incorporation after 96 h of treatment. However, [3H]thymidine incorporation into DNA after 24 h of treatment, with injection of a combination of isoproterenol/alpha-lactalbumin or isoproterenol/trifluoperazine, was similar to injections of isoproterenol alone; suggesting that acinar cells could be stimulated to undergo a single round of DNA synthesis. Northern blot analysis of myc and fos expression followed a similar pattern of down-regulation to control levels after 96 h but not after 24 h. Hybridization with erb B showed little change with proliferation, confirming previous observations on protein levels of the EGF-receptor in acinar cells. Western blot analysis of nuclear protein expression of myc revealed that isoproterenol caused an increase in a 62-kDa protein which was again down-regulated with inhibition of cell proliferation. Analysis of protein levels of Rb110 protein showed no change in protein level in the nucleus with cell proliferation, but did show an associated increase in protein phosphorylation in response to growth stimulation.     

Activation of macrophages by peroxidases

Peritoneal macrophages from C57BL/6 mice were activated in vitro with various peroxidases and their cytotoxic activity toward 3T12 cells was determined. Destruction of 3T12 cells by macrophages stimulated with horseradish peroxidase, lactoperoxidase, and microperoxidase was observed at peroxidase concentrations as low as 9, 1.6, and 200 nM, respectively. A 50% cytotoxic effect was obtained at peroxidase concentrations of 0.9, 1.6, and 1.5 microM, respectively. The macrophage-stimulating activity of horseradish peroxidase was not destroyed by boiling. This, together with the high activity of microperoxidase, indicates that the macrophage-stimulating activity of the peroxidases is probably associated with the heme portion of the enzymes. On a molar basis the peroxidases are much less potent macrophage activators than interferon (alpha + beta) and endotoxin. Nevertheless, our data clearly indicate that peroxidases are a group of enzymes capable of inducing macrophage activation, resulting in cytostatic and/or cytocidal activity.     

The use of lead as an ionic tracer for investigating routes of passive fluid transfer

Several inorganic ions, including lead, barium, silver, and thallium, have been tested as possible tracers for demonstrating fluid-accessible channels in functional epithelia at the ultrastructural level. The most useful of the ionic tracers examined was the lead (plumbous) ion, administered for short time intervals (less than 2 min) and "captured" with phosphate used as the buffer in the fixative. Passive fluid and ion-accessible channels of rat parotid salivary gland have been examined with this method. At short tracer infusion times (0.5-1.0 min), localization of the tracer was primarily extracellular, although intracellular deposits were observed in the following sites: smooth membrane-delimited endocytic vesicles of both epithelial and connective tissue cells, inner Golgi cisternae, and occasional cisternae of rough endoplastic reticulum. The lead tracer readily penetrated tight junctions between parotid acinar cells but rarely passed through the tight junctions between intercalated duct cells and did not penetrate junctions of striated duct cells. Fat cells observed in the stroma of this gland were the only cells that exhibited lead tracer in the cytosol, suggesting that the plasmalemma of this cell type is more permeable to exogenous ions than the plasmalemma of other cell types present in this gland.     

Immunolocalization of electroneutral Na(+)-HCO cotransporters in human and rat salivary glands

Patterns of salivary HCO secretion vary widely among species and among individual glands. In particular, virtually nothing is known about the molecular identity of the HCO transporters involved in human salivary secretion. We have therefore examined the distribution of several known members of the Na(+)-HCO cotransporter (NBC) family in the parotid and submandibular glands. By use of a combination of RT-PCR and immunoblotting analyses, the electroneutral cotransporters NBC3 and NBCn1 mRNA and protein expression were detected in both human and rat tissues. Immunohistochemistry demonstrated that NBC3 was present at the apical membranes of acinar and duct cells in both human and rat parotid and submandibular glands. NBCn1 was strongly expressed at the basolateral membrane of striated duct cells but not in the acinar cells in the human salivary glands, whereas little or no NBCn1 labeling was observed in the rat salivary glands. The presence of NBCn1 at the basolateral membrane of human striated duct cells suggests that it may contribute to ductal HCO secretion. In contrast, the expression of NBC3 at the apical membranes of acinar and duct cells in both human and rat salivary glands indicates a possible role of this isoform in HCO salvage under resting conditions.     

Tissue distribution of constitutive and induced soluble peroxidase in rat. Purification and characterization from lacrimal gland.

A thorough search for a soluble peroxidase in 31 different tissues of rat indicated the presence of a constitutive activity only in lacrimal, preputial and submaxillary gland. An induced soluble peroxidase activity was also detected in the lactating mammary gland and in the estrogen-induced uterine secretory fluid. The lacrimal gland was the richest source of the enzyme. No peroxidase activity was detected in the lactating mammary gland of mouse and hamster nor in the preputial gland of mouse and uterine fluid of hamster. The three constitutive and two induced soluble peroxidases of rat had a native molecular mass of 73 kDa by gel filtration and they showed a similar mobility in native PAGE. Lactoperoxidase of cow's milk and solubilized rat membrane-bound peroxidases of uterus, intestine and bone marrow showed in native PAGE a mobility which was distinctly different from that of rat soluble peroxidases. As the lacrimal gland of rat was the richest source of soluble peroxidase, the enzyme was purified from this gland to apparent homogeneity; SDS/PAGE then showed a single band of molecular mass 75 kDa which was similar to that obtained by gel filtration. Peroxidase also purified from preputial and submaxillary gland, as well as commercial lactoperoxidase, had a similar molecular mass on SDS/PAGE to purified lacrimal peroxidase. The visible spectrum of lacrimal peroxidase was similar to that of lactoperoxidase but different from membrane-bound peroxidase of rat neutrophils. On isoelectric focussing, purified lacrimal peroxidase resolved into about 14 multiple forms spanning a pI range of 6.5-3.5 while lactoperoxidase focussed at the cathode. Evidence presented suggests that the multiple forms are possibly due to differences in glycosylation. Immunodiffusion, immunoprecipitation and Western blot using antilacrimal peroxidase serum showed a similar interacting species for all five soluble peroxidases of rat while membrane-bound peroxidases showed no interaction. Although in immunodiffusion, the antiserum failed to cross-react with lactoperoxidase it did interact with lactoperoxidase on Western blot. The results indicate that the various constitutive and induced soluble peroxidases of rat tissues are similar to lacrimal peroxidase but are distinctly different from the known membrane-bound peroxidases of rat. However the lacrimal peroxidase shows both similarities as well as dissimilarities with bovine lactoperoxidase. This soluble peroxidase system of rat could be useful to study tissue-specific regulation of gene expression at the molecular level.     

Isoproterenol-stimulated labelling of particulate proteins by using [adenylate-32P]NAD+ independent on a cAMP-dependent protein kinase in parotid acinar cells.

When saponin-permeabilized rat parotid acinar cells were incubated with [adenylate-32P]NAD+, labelling of proteins (33, 27 and 23 kDa) in particulate fractions of the cells was stimulated by isoproterenol. The effect of isoproterenol was completely blocked by a beta-antagonist. Both forskolin or cAMP mimicked the effect of isoproterenol on the labelling. However, an inhibitor of cAMPdPK failed to induce complete inhibition of the effects of isoproterenol, forskolin and cAMP. When the labelled proteins were treated with snake venom phosphodiesterase, neither [32P]5'-AMP nor [32P]phosphoribosyladenosine was released. These results suggest that covalent modification of proteins with NAD+, which is distinct from ADP-ribosylation and cAMPdPK-dependent phosphorylation, is coupled to beta-receptor-cAMP signalling system in rat parotid acinar cells.