The effects of vinblastine on acinar cells of the exorbital lacrimal gland of the rat

Summary The effects of vinblastine treatment on acinar cells of the rat exorbital lacrimal gland were studied by electron microscopy. Experimental animals of both sexes were given single intraperitoneal injections of (1) vinblastine (4mg/kg body weight) at 1 to 24 h before sacrifice; (2) pilocarpine (20 mg/kg b.w.) for 1 h; or (3) vinblastine for l h followed by pilocarpine for 1 h.Vinblastine treatment caused a number of changes including autophagocytosis, formation of intracisternal granules, and alteration of secretory granules. These changes varied in extent and onset between male and female rats. In addition, the Golgi apparatus was reduced in size and dispersed throughout the cytoplasm. Mitotic figures were commonly observed. Moreover, vinblastine inhibited the pilocarpine-stimulated degranulation of the acinar cells.In view of the known anti-microtubular action of vinblastine, these results suggest that microtubules are involved in various aspects of the transport, packaging, and secretion of exportable proteins in the lacrimal gland. Additionally, autophagocytosis and alteration of secretory granules may partially result from the interaction of vinblastine with membranes.The authors thank Mr. Steve Coriell and Mr. Steve Floyd for preparing the micrographs. Robert Kelly also thanks Dr. George Chapman for his support during the initial phase of this project.     

Androgen regulation of secretory component synthesis by lacrimal gland acinar cells in vitro

This study sought to determine whether androgens directly stimulate the production of secretory component (SC) by acinar cells from the rat lacrimal gland. Homogeneous populations of acrinar cells were isolated from lacrimal tissues by serial enzymatic digestion and Ficoll gradient centrifugation and then cultured on reconstituted basement membranes in supplemented, serum-free medium. Acinar cell exposure in vitro to dihydrotestosterone (DHT) resulted in a significant increase in cellular SC output. This hormone action was dose dependent and androgen specific. Testosterone, but not 17 beta-estradiol, progesterone, dexamethasone, or aldosterone, also induced a considerable elevation in acinar cell SC production. The effect of testosterone may not require intracellular enzymatic conversion to DHT. The impact of androgens on SC output was associated with enhanced cellular synthesis and secretion and did not involve variations in acinar cell viability or density. Moreover, the SC response to DHT occurred irrespective of whether lacrimal gland acinar cells were obtained from young adult male or female rats. In contrast, the androgen-related rise in SC production was significantly reduced in acinar cells isolated from tissue of orchiectomized and hypophysectomized rats. In summary, these findings demonstrate that androgens directly increase the synthesis of SC by lacrimal gland acinar cells in vitro. This effect, however, may be significantly altered by prior changes in the endocrine environment of acinar cells in vivo.     

Harderianization is another sexual dimorphism of rat exorbital lacrimal gland

The exorbital lacrimal glands (ELG) of rats were examined for both sexes to determine what degree of harderianization occurred as a function of age and after castration, and to investigate its time course and origin in ELG. Light microscopically, very small Harderian foci were seen in the ELG of both sexes at 3 weeks of age. As the male rats became older, the relative volume of the Harderian gland (HG) cells in the ELG increased. At age 6 months, the value was 1.25 +/- 0.31% in males and 0.13 +/- 0.05% in females (p less than 0.05). After castration, a significant decrease (0.21 +/- 0.01%, p less than 0.05) was observed in that of male ELG. In contrast, in female ELG, HG cells were inconspicuous and the relative volume of those did not vary during this experimental period or after castration. It appeared that the HG cells had developed from undifferentiated basal cells of the acini and the intercalated ducts in the ELG at age 2-6 months. Then, at age 22 months, they also probably developed from those of the excretory ducts of the ELG.     

Demonstration and characterization of cytosol androgen receptor in rat exorbital lacrimal gland

The presence of a macromolecule which binds androgen with a high affinity and a low capacity was demonstrated in the cytosol of the lacrimal glands of male and female rats. Evidence was found that this macromolecule was a protein by treatment with protease, trypsin or heat. A specific 8-8.5 S peak was obtained in both sexes by glycerol gradient centrifugation in low salt condition, whereas a specific 5.2 S peak was found in high salt condition. This protein could bind to DNA-cellulose after treatment of androgen-cytosol complexes by warming (25 degrees C 15 min) or exposure under high salt (0.4 M KCl). These results suggested that this protein was an androgen receptor.     

Endocrine, neural, and immune control of secretory component output by lacrimal gland acinar cells.

Androgens regulate the synthesis and secretion of secretory component (SC), the IgA antibody receptor, by acinar cells from the lacrimal gland. However, this hormone action may be susceptible to significant modification by other agents from the endocrine, nervous, or immune systems. To investigate the nature of this neuroimmunoendocrine interaction, the present study examined the impact of hormones, neurotransmitters, and lymphokines on basal and androgen-induced SC production by lacrimal gland acinar cells in vitro. Our results demonstrated that vasoactive intestinal peptide, the beta-adrenergic agonist, isoproterenol, PGE2, IL-1 alpha, IL-1 beta, and TNF-alpha significantly increased media SC levels in control or androgen-containing cell cultures. In contrast, the cholinergic agonist, carbachol, significantly decreased cellular SC output. These effects may be mediated through the agents' known capacity to alter intracellular cAMP levels. In support of this hypothesis, exposure of acinar cells to stimulators or analogues of cAMP resulted in a significant enhancement of SC production. Thus, these findings indicate that SC output in lacrimal tissue may be modulated by interactions between the endocrine, nervous and immune systems.     

Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland

Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b(-/-) and Rab27(ash/ash)/Rab27b(-/-) mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release.     

Formation of secretion granules in the Golgi apparatus of pancreatic acinar cells of the rat

The three-dimensional structure of the Golgi apparatus and its components has been analyzed in sections of pancreatic acinar cells by using stereopairs of electron microscope photographs. Pancreatic tissue fixed in glutaraldehyde was postfixed in reduced osmium, and the sections were stained with lead citrate. Tissues were also treated to demonstrate phosphatase activity (i.e., nicotinamide adenine dinucleotide phosphatase, NADPase; thiamine pyrophosphatase, TPPase; cytidine monophosphatase, CMPase). The following stacked components were observed along the branching, anastomotic, continuous, ribbonlike Golgi apparatus. 1) On the cis-face of the Golgi stack there was a tubular membranous network known to be osmiophilic and referred to as the cis-osmiophilic tubular network or cis-element. 2) A first, poorly fenestrated saccule, unreactive for the phosphatases tested, was slightly distended in places and contained a fluffy granulofilamentous material. 3) The subjacent three or four saccules, reactive for NADPase and/or TPPase, showed dilated portions containing a granulofilamentous secretory material similar to that filling the rest of the saccule. They also showed nondilated portions perforated with large fenestrations, some of which were in register and formed wells containing 80-nm vesicles. The dilated portions of these saccules were present at random along the length of the saccules and were not located exclusively at their edges. 4) The remaining one or two elements of the stack, CMPase positive, showed dilated spheroidal portions or prosecretory granules containing a homogeneous secretory material and flattened fenestrated regions free of secretory material and having the appearance of networks of narrow membranous tubules. 5) Lastly on the trans-aspect of the stack there were detached prosecretory granules reactive for CMPase and surrounded by a corona of small vesicles, and smooth-surfaced spherical CMPase-negative granules having a denser content that were identified as fully formed secretion granules; there were also occasional free trans-tubular networks strongly reactive for CMPase that appeared to undergo fragmentation and numerous small vesicles free from acid-phosphatase activity. These various images were interpreted as indicating that prosecretory granules formed in relation to two or three fenestrated saccules on the trans-side of the stack. Such granules, following their detachment from the trans-face of the stack, their separation from trans-tubular networks, and condensation of their content, yielded mature secretion granules.     

Calcium dependence of exocytosis in lacrimal gland acinar cells

Simultaneous measurements of membranecapacitance and intracellular calcium concentration were used toexamine the calcium dependence of exocytosis in single acinar cellsfrom mouse lacrimal gland and to establish the quantitative relationbetween calcium concentration and rate of exocytosis. Application ofadrenergic or muscarinic agonists elevated intracellular calcium andevoked exocytosis, as indicated by an increase in membrane capacitance of single cells. The capacitance response to agonist stimulation waseliminated by internal dialysis with the calcium buffer EGTA, whichdemonstrated that the increase in intracellular calcium was necessaryfor agonist-evoked exocytosis. When internal calcium was elevated byapplication of the calcium ionophore ionomycin, exocytosis was evokedin the absence of agonist stimulation. Thus an increase inintracellular calcium was necessary and sufficient for exocytosis insingle acinar cells. The rate of change of membrane capacitanceincreased as approximately the third power of the calciumconcentration, which is similar to the dependence of exocytosis rate oncalcium concentration in other secretory 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.     

Direct interaction between Rab3D and the polymeric immunoglobulin receptor and trafficking through regulated secretory vesicles in lacrimal gland acinar cells

The lacrimal gland is responsible for tear production, and a major protein found in tears is secretory component (SC), the proteolytically cleaved fragment of the extracellular domain of the polymeric Ig receptor (pIgR), which is the receptor mediating the basal-to-apical transcytosis of polymeric immunoglobulins across epithelial cells. Immunofluorescent labeling of rabbit lacrimal gland acinar cells (LGACs) revealed that the small GTPase Rab3D, a regulated secretory vesicle marker, and the pIgR are colocalized in subapical membrane vesicles. In addition, the secretion of SC from primary cultures of LGACs was stimulated by the cholinergic agonist carbachol (CCH), and its release rate was very similar to that of other regulated secretory proteins in LGACs. In pull-down assays from resting LGACs, recombinant wild-type Rab3D (Rab3DWT) or the GDP-locked mutant Rab3DT36N both pulled down pIgR, but the GTP-locked mutant Rab3DQ81L did not. When the pull-down assays were performed in the presence of guanosine-5'-(gamma-thio)-triphosphate, GTP, or guanosine-5'-O-(2-thiodiphosphate), binding of Rab3DWT to pIgR was inhibited. In blot overlays, recombinant Rab3DWT bound to immunoprecipitated pIgR, suggesting that Rab3D and pIgR may interact directly. Adenovirus-mediated overexpression of mutant Rab3DT36N in LGACs inhibited CCH-stimulated SC release, and, in CCH-stimulated LGACs, pull down of pIgR with Rab3DWT and colocalization of pIgR with endogenous Rab3D were decreased relative to resting cells, suggesting that the pIgR-Rab3D interaction may be modulated by secretagogues. These data suggest that the novel localization of pIgR to the regulated secretory pathway of LGACs and its secretion therefrom may be affected by its novel interaction with Rab3D.     

Effects of secretory stimulation on the Golgi apparatus and GERL of rat parotid acinar cells

The structure and cytochemistry of the Golgi apparatus and GERL of rat parotid acinar cells was studied after in vivo secretory stimulation with isoproterenol. Discharge of mature secretory granules was complete within 1 hr after isoproterenol injection, but immature granules in the Golgi region or near the lumen were not released. At early times (1-5 hr) after isoproterenol, acid phosphatase (AcPase) activity was markedly increased in GERL and immature secretory granules compared to uninjected controls. GERL appeared increased in extent and numerous continuities with immature granules were observed. Reaccumulation of mature secretory granules was first evident at 5 hr, and was almost complete by 16 hr after isoproterenol. Thiamine pyrophosphatase (TPPase) activity, normally restricted to the trans Golgi saccules, was frequently present in immature granules during this time. Narrow cisternae resembling GERL, occasionally in continuity with immature granules, also contained TPPase reaction product. By 16-24 hr after stimulation, the activity and distribution of AcPase and TPPase were similar to control cells. These results demonstrate the dynamic nature of the Golgi apparatus and GERL in parotid acinar cells, and emphasize the close structural and functional relationship between these two structures.     

Leucine aminopeptidase (LAP) activity and sexual dimorphism in rat exorbital lacrimal gland

Leucine amonopeptidase (LAP) activity was histochemically studied in the rat exorbital lacrimal gland. The enzyme is present in the secreting cells of both male and female prepuberal rats, in vivo and in monolayer cultures, while in the adult rat it is demonstrable only in the female. Furthermore, LAP is influenced by the sex hormones, disappearing in the female gland after testosterone treatment and appearing in the adult male gland after administration of estradiol or cyproterone acetate. These results show that the behaviour of LAP can be considered as another character of sexual dimorphism of the rat exorbital gland. Furthermore, our findings, showing an inverse relationship between LAP activity and the PAS positivity of the secretion products, suggest the hypothesis that the presence of this exopeptidase could induce qualitative modifications of one or more secreted glycoprotein.     

Immunolocalization of myoepithelial cells in isolated acini of rat exorbital lacrimal gland: Cellular distribution of muscarinic receptors

The secretion of proteins and fluids from the exorbital lacrymal gland of rat is mainly controlled by muscarinic receptors. In a recent pharmacological study, Mauduit et al (Am J Physiol (1993) 264, C1550–C1560) identified a homogeneous population of M₃ muscarinic receptors in preparations of acini from these tissues. In order to define the cellular composition of these acini and localize the muscarinic receptors; we have performed an immunofluorescent labelling study combined with confocal scanning microscopy. Antibodies raised against components of the different cytoskeletal networks (α-smooth muscle actin, cytokeratin peptide 14 and α-tubulin) revealed the presence of two different cell types. Cells with a stellate form are identified as myoepithelial cells, whereas rounded cells are secretory acinar cells. Both cell types are reactive with an antibody specifically directed against the muscarinic receptor. However, myoepithelial cells appear more intensely labelled than acinar cells. The roles of myoepithelial cells and secretory cells in the physiological function of the gland are discussed in terms of the distribution of muscarinic receptors.     

Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells

During pregnancy, lymphocytes infiltrating the rabbit lacrimal gland disperse to the interacinar space from their normal focal concentrations, basal fluid secretion decreases, pilocarpine-induced fluid secretion increases, and stimulated fluid protein concentration decreases. Ductal epithelial cell prolactin (PRL) content increases and redistributes from the apical to the basal-lateral cytoplasm. A replication-incompetent adenovirus vector for rabbit PRL (AdPRL) was used to test the hypothesis that increased intracrine/autocrine PRL signaling alters secretory protein traffic in an ex vivo lacrimal acinar cell model. AdPRL had no discernable influence on microtubules or actin microfilaments or their responses to carbachol (CCh). Endogenous and transduced PRLs exhibited similar, nonpolarized, punctate distributions. Cells secreted PRL consititutively and at increased rates in response to CCh. In contrast, constitutive secretion of beta-hexosaminidase was negligible, suggesting that the constitutive pathway for PRL is relatively inaccessible to typical secretory proteins. AdPRL had no significant effect on total secretion of beta-hexosaminidase or syncollin-green fluorescent protein (GFP), a chimeric secretory protein construct. However, it reversed the polarized distributions of vesicles containing rab3D and syncollin-GFP. Live-cell imaging indicated that AdPRL redirected CCh-dependent syncollin-GFP exocytosis from the apical plasma membrane to the basal-lateral membrane. Elevated concentrations of exogenous rabbit PRL in the ambient medium elicited similar changes. These observations suggest that elevated PRL, as occurs in the physiological hyperprolactinemia of pregnancy, induces lacrimal epithelial cells to express a mixed exocrine/endocrine phenotype that secretes fluid to the acinus-duct lumen but secretes proteins to the underlying tissue space. This phenotype may contribute to the pregnancy-associated immunoarchitecture.     

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.