Growth of Purified Lacrimal Acinar Cells in Matrigel Raft Cultures

Schechter, Joel; Stevenson, Douglas; Chang, Donald C.; Chang, Natalie; Pidgeon, Michael; Nakamura, T.; Okamoto, Curtis T.; Trousdale, Melvin D.; Mircheff, Austin K.

doi:10.1006/exer.2001.1158

Cited by 34 publications

(33 citation statements)

References 17 publications

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“…They were used in accordance with the Guiding Principles for the Use of Animals in Research, with a protocol approved by the Institutional Animal Care and Use Committee. Lacrimal gland acinar cells were isolated and cultured as described previously (13,14,38). For biochemical and functional studies the lacrimal acinar cells from 4-kg rabbits were resuspended at 4°C in HSM containing 10% FBS, 5 ng/ml EGF, and 1 mg/ml Matrigel and then warmed to 37°C and incubated for 1 h to promote formation of Matrigel particles, referred to as "rafts" (38).…”

Section: Methodsmentioning

confidence: 99%

Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells

Wang¹,

Chiu²,

Nakamura³

et al. 2007

American Journal of Physiology-Endocrinology and Metabolism

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-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 ␤-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 ␤-hexosaminidase or syncollingreen 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. ocular surface; mucosal immunity; pregnancy; Sjögren's syndrome THE OCULAR SURFACE SYSTEM maintains a thin fluid film that comprises an external milieu necessary for optimal homeostasis of its mucosal tissues and of the cornea. Disruptions of ocular surface homeostasis frequently elicit sensations of dryness, itching, or burning. These symptoms may be associated with immune system diseases, such as Sjögren's syndrome and graft vs. host disease, but most cases occur without the typical signs of autoimmune disease. They occur more frequently in women than men and are often associated with altered states of the systemic hormonal milieu: perimenopause, postmenopause, pregnancy, lactation, oral contraceptive use (39), and estrogen or estrogen-progesterone replacement therapy (35,36). Among men, the frequency increases with age (8).Aging in men is accompanied by gradual decreases in gonadal and adrenal production of androgens. The physiological states associated with altered ocular surface system homeostasis in women share one well-known common denominator, a decrease in the amount of testosterone that...

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Section: Methodsmentioning

confidence: 99%

Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells

Wang¹,

Chiu²,

Nakamura³

et al. 2007

American Journal of Physiology-Endocrinology and Metabolism

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“…At the same time, vehicle or 100 mM carbachol, respectively, was added to the remaining groups, which were incubated for another 20 m, then centrifuged. In a second series of experiments, acinar cells were established in Matrigel rafts as described by Schechter et al [29]. After 2 days, rafts were transferred to fresh medium lacking fetal bovine serum and epidermal growth factor (EGF).…”

Section: Methodsmentioning

confidence: 99%

“…Specific binding was calculated as the component of the total binding displaced by 2.5 mM atropine. Use of antibodies for Western blot detection of G 11 , G q , G i3 and G s [19], VAMP2 and rab3D [25] and pIgR [29] has been described in previous publications. The antibody for protein kinase Ca (PKCa) detected a single band.…”

Section: Methodsmentioning

confidence: 99%

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Biochemical Changes Contributing to Functional Quiescence in Lacrimal Gland Acinar Cells after Chronic Ex Vivo Exposure to a Muscarinic Agonist

Wang

Xie

et al. 2003

Scand J Immunol

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Profound secretory dysfunction can be associated with relatively modest lymphocytic infiltration of the lacrimal and salivary glands of Sjögren's syndrome (SjS) patients. SjS patients' sera contain autoantibodies to M3 muscarinic acetylcholine receptors (MAChR) that have variously been reported to have agonistic and antagonistic effects. We sought to identify consequences of chronic agonist stimulation by maintaining acinar cells from rabbit lacrimal glands for 20 h in the presence or absence of 10 mM carbachol (CCh). Exposure to CCh diminished the cells' ability to elevate cytosolic Ca 2þ and secrete b-hexosaminidase in response to acute stimulation with 100 mM CCh, but it enhanced their secretory responses to phenylephrine and ionomycin. Secretory vesicles appeared normal by electron microscopy, but confocal fluorescence microscopy revealed depletion of the secretory vesicle membrane marker, rab3D, and decreased ability to recruit secretory transport vesicles in response to acute 100 mM CCh. Additionally, the apical cortical actin cytoskeleton was disrupted and diminished compared to the basal-lateral cortical network. Subcellular fractionation analyses revealed that total membrane phase protein content was increased. The contents of b-hexosaminidase and MAChR relative to total protein were not significantly altered, and MAChR abundance in the plasma membrane fraction was increased as the result of redistribution from endomembrane pools. However, relative cellular contents of the heterotrimeric guanosine triphosphate (GTP)-binding proteins, G q and G 11 , were decreased. Additional biochemical changes included decreased contents of 47 kDa G s and G i3 , protein kinase Ca and rab3D and polymeric immunoglobulin (Ig) receptors; internalization of Na,K-ATPase from the plasma membranes to endomembrane compartments and decreased content of b-hexosaminidase in the lysosomes. The observations demonstrate that chronic exposure to a MAChR agonist induces refractoriness to optimal stimulation, without causing receptor downregulation, by downregulating postreceptor-signalling mediators and effectors. The cells' secretory mechanisms for IgA and electrolytes also appear to be impaired, as does their ability to properly sort proteins to the lysosomes.

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“…Animals were sacrificed by cervical dislocation and/or with carbon dioxide. Exorbital lacrimal glands were removed and some tissue fragments were fixed and processed for epoxy embedment for electron microscopy (EM) as previously described (Schechter et al, 2002). Additional fragments of lacrimal glands were fixed in 4% paraformaldehyde/4% sucrose in Dulbecco's phosphate buffered saline (DPBS) at room temperature for 3-4 hours and then transferred to 30% sucrose in DPBS at 4°C overnight.…”

Section: Isolation and Processing Of Mouse Lacrimal Glandsmentioning

confidence: 99%

Male NOD mouse external lacrimal glands exhibit profound changes in the exocytotic pathway early in postnatal development

Costa

Veigh

et al. 2006

Experimental Eye Research

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The lacrimal glands of male NOD mice exhibit many of the features of the human lacrimal gland in patients afflicted with the autoimmune disease, Sjögren's syndrome, including loss of secretory functions and lymphocytic infiltration into the lacrimal gland. To elucidate the early changes in the secretory pathway associated with development of Sjögren's syndrome, we investigated the organization of the exocytotic pathway in lacrimal glands of age-matched male BALB/c and NOD mice. Cryosections from lacrimal glands from 1 and 4 month male BALB/c and NOD mice were processed for confocal fluorescence and electron microscopic evaluation of different participants in exocytosis. No changes in apical actin filaments were noted in glands from NOD mice, but these glands exhibited thickening of basolateral actin relative to that seen in the BALB/c mice. Rab3D immunofluorescence associated with mature secretory vesicles was distributed abundantly in a continuous vesicular network concentrated beneath the apical plasma membrane in glands from 1 and 4 month BALB/c mice. In glands from 1 month NOD mice, rab3D immunofluorescence exhibited marked discontinuity and irregularity in the vesicular labeling pattern. While this change was also detected in glands from 4 month NOD mice, many of these glands exhibited an additional extension of rab3D labeling through the cell to the basolateral membrane. Electron microscopic analysis confirmed the formation of irregularly-shaped, unusually large secretory vesicles in lacrimal glands from NOD mice. Quantitation of multiple secretory vesicles from electron micrographs revealed a significant (p ≤5) increase in the percentage of secretory vesicles incorporated into multivesicular aggregates in lacrimal glands from 1 and 4 month NOD mice compared to BALB/c mice. The M3 muscarinic receptor, a key signaling effector of exocytosis, was redistributed away from its normally basolateral locale in glands from BALB/c mice, with concomitant enrichment in intracellular aggregates in glands from NOD mice. These findings show that lacrimal glands in NOD mice as young as 1 month contain aberrant secretory vesicles with altered effector composition that undergo premature cytoplasmic fusion, and that changes in the distribution of the M3 muscarinic receptor occur within the same time frame.

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Growth of Purified Lacrimal Acinar Cells in Matrigel Raft Cultures

Cited by 34 publications

References 17 publications

Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells

Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells

Biochemical Changes Contributing to Functional Quiescence in Lacrimal Gland Acinar Cells after Chronic Ex Vivo Exposure to a Muscarinic Agonist

Male NOD mouse external lacrimal glands exhibit profound changes in the exocytotic pathway early in postnatal development

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