Characterization of β-hexosaminidase secretion in rabbit lacrimal gland

Andersson, Sofia V.; Edman, Maria C.; Bekmezian, Arpi; Holmberg, Jens; Mircheff, Austin K.; Gierow, J. Peter

doi:10.1016/j.exer.2006.05.013

Cited by 16 publications

(17 citation statements)

References 25 publications

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“…The following antibodies and dilutions were used for Western blotting: phosphorylated p38 LG single cell isolation and stimulation LG single acinar cells were isolated and cultured as previously described [6]. Cells were seeded in 48-well plates for secretion assay (6.4 x 10 5 cells/well) and 6-well plates for lysate preparation in Western blotting experiments (6.4 x 10 6 cells/well).…”

Section: Antibodiesmentioning

confidence: 99%

“…Supernatants were then centrifuged at 13,000 x g for 5 min and the aspirated supernatants were stored at -20°C. Secretion was measured using a marker for secretion, β-hexosaminidase [6]; the methodology is described elsewhere [9]. In brief, a substrate for β-hexosaminidase was added to the supernatant, yielding a fluorescent product which was measured in a Flurolog 3-22 or Fluoromax 3 fluorometer (Horiba Jobin Yvon, Edison, NJ) with excitation at 365 nm and emission at 460 nm.…”

Section: In Vitro Secretion Assaymentioning

confidence: 99%

“…Cells were seeded in 48-well plates for secretion assay (6.4 x 10 5 cells/well) and 6-well plates for lysate preparation in Western blotting experiments (6.4 x 10 6 cells/well). The cells were cultured for 2-3 days in order for them to reorganize into acini-like structures [6] and then washed twice in Hank's culture medium complemented with 10 mM HEPES and 1 mM calcium chloride (all from Sigma-Aldrich, St Louis, MO).…”

Section: Antibodiesmentioning

confidence: 99%

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p38 Mitogen-activated protein kinase modulates exocrine secretion in rabbit lacrimal gland

Carlsson

Gierow

2012

Cellular and Molecular Biology Letters

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Abstract:The lacrimal gland (LG) is an exocrine gland important for secretion of the tear film. The kinase p38 has important signal transduction functions, e.g. in gene transcription, but has previously not been known to modulate exocrine secretion. The aim of the current study was to investigate the role of p38 in carbachol (Cch)-induced LG secretion in LG acinar cells in vitro. Western blotting was used to determine the phosphorylation status of p38 and p42/44 and determine expression of p38 isoforms. To determine the effect of p38 inhibition on LG secretion, PD 169316, a general p38 inhibitor, and SB 239063, an inhibitor of p38α and β, were added to the cells prior to secretion measurements. The results revealed activation of p38 mediated by Cch stimulation and inhibition of Cch-induced secretion as a result of p38 inhibition. The inhibition was observed with PD 169316 isoforms, but not with SB 239063. The p38δ isoform was shown to have robust expression both by Western blotting of acinar cells and immunofluorescence of the whole gland. In conclusion, p38 activation mediates secretion in cholinergic stimulation of rabbit LG cells.

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Section: Antibodiesmentioning

confidence: 99%

Section: In Vitro Secretion Assaymentioning

confidence: 99%

Section: Antibodiesmentioning

confidence: 99%

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p38 Mitogen-activated protein kinase modulates exocrine secretion in rabbit lacrimal gland

Carlsson

Gierow

2012

Cellular and Molecular Biology Letters

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“…The difficulty of this procedure not only pertains to cell isolation but also to obtaining good viability (higher than 80% of the total number of cells) to ensure a large population of target cells with preserved secretory activity (17) . Previous studies have focused on the aspects of LGACs in primary culture, applying standard amounts of insulin in the culture media (12,18) . The aims of this study were to determine the optimal conditions for establishing a line of primary LGACs in culture and to study the effect of insulin in variable concentrations in the culture medium on acinar cell growth, secretory function, and viability.…”

Section: Introductionmentioning

confidence: 99%

“…Several attempts to enhance the growth of acinar cells in culture have been made, but after a span of approximately 3 weeks, LGACs exhibited decreased viability, with high numbers of apoptotic cells (11)(12)(13)(14) . Exocrine acinar cells are fragile, post-mitotic epithelial cells with marked polarity.…”

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confidence: 99%

Lacrimal gland primary acinar cell culture: the role of insulin

Malki

Dias

Jorge

et al. 2016

Arquivos Brasileiros De Oftalmologia

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The condition of lacrimal glands (LG) is influenced by components of the immune, neural, and endocrine systems (1)(2)(3)(4)(5) . Insulin is thought to be of major importance, based on observations related to its deprivation in diabetes mellitus (DM), on LG, tear film, and ocular surface in humans, animal models, and in culture (6)(7)(8) . Lacrimal gland acinar cell (LGAC) culture is an appropriate model for improving the understanding of the influence of insulin on these cells.In previous studies, the culture of LGACs from different species was performed over an average period of 21 days (9,10) . Several attempts to enhance the growth of acinar cells in culture have been made, but after a span of approximately 3 weeks, LGACs exhibited decreased viability, with high numbers of apoptotic cells (11)(12)(13)(14) . Exocrine acinar cells are fragile, post-mitotic epithelial cells with marked polarity. This indicates that the apical and basal sides are clearly positioned but also that intracellular organelles are located at one or the other pole, depending on their cellular functions. Thus, an adequate extracellular matrix and other specific ingredients are needed in the culture media to mimic in vivo conditions (15,16) . The handling during isolation and culture procedure of LGACs should be gentle ABSTRACT Purpose: The goal of the present study was to establish a protocol for primary culture of lacrimal gland acinar cells (LGACs) and to assess the effect of adding insulin to the culture media. Methods:LGACs were isolated and cultured from lacrimal glands of Wistar male rats. The study outcomes included cell number, viability, and peroxidase release over time and in response to three concentrations of insulin (0.5, 5.0, and 50.0 μg/mL). Results: In LGAC primary culture, cells started to form clusters by day 3. There was a time-response pattern of peroxidase release, which rose by day 6, in response to carbachol. Culture viability lasted for 12 days. An insulin concentration of 5.0 μg/mL in the culture medium resulted in higher viability and secretory capacity. Conclusions:The present method simplifies the isolation and culture of LGACs. The data confirmed the relevance of adding insulin to maintain LGACs in culture. Keywords

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Traffic of endogenous, transduced, and endocytosed prolactin in rabbit lacrimal acinar cells

Wang

Chiu

Nakamura

et al. 2007

Experimental Eye Research

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The rabbit lacrimal gland undergoes an immunophysiological transformation during pregnancy, reminiscent of that of the mammary gland as it prepares to deliver secretory IgA into the nascent fluid product. The contents of TGF-beta and prolactin (PRL) within ductal epithelial cells increase, and their primary localizations shift from the apical to the basal cytoplasm, suggesting a transformation from exocrine to paracrine secretion. Studies with ex vivo acinar cell models demonstrated that elevated PRL suppresses traffic of secretory proteins into the regulated exocrine apparatus and directs them into a novel, induced, regulated paracrine apparatus [Wang, Y., Chiu, C.T., Nakamura, T., Walker, A.M., Petridou, B., Trousdale M.D., Hamm-Alvarez S.F., Schechter J.E., Mircheff A.K., 2007. Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells. Am. J. Physiol. Endocrinol. Metab. 292, E1122-E1134]. However, it was not clear whether PRL itself entered the induced paracrine apparatus. In the present study, confocal immunofluorescence microscopy revealed that natively expressed PRL and over-expressed PRL co-localized with PRL receptors (PRLR); rab11, a marker for the recycling endosome; gamma-adaptin, a marker for the Golgi complex and trans-Golgi network; and rab7, a marker for the autophagic lysosomal apparatus. Natively expressed, over-expressed, and endocytosed PRL also co-localized with rab4 and rab5A, markers for the early endosome, and with rab3D, a marker for regulated exocrine secretory vesicles. Endocytosed PRL was stored in intact form and released in response to stimulation with carbachol. Subcellular fractionation analysis detected relative excesses of PRL over PRLR in fractions that contained fragments of the recycling endosome and fractions that contained both secretory vesicle fragments and prelysosomal and autolysosomal fragments. EM-gold microscopy demonstrated PRL within small vesicles, consistent with endosomes or secondary lysosomes, and in large vesicles, consistent with regulated secretory vesicles. The secretory vesicles were preponderantly localized in the apical cytoplasm of control cells, and in the basal cytoplasm of PRL over-expressing cells. These results indicate that when lacrimal epithelial cells synthesize PRL, and when they endocytose it from their ambient medium, they traffic it both into the endosomes that constitute the constitutive transcytotic paracrine apparatus and also into regulated secretory vesicles, which are associated with the exocrine apparatus at low PRL levels and with the induced paracrine apparatus at high PRL levels.

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Characterization of β-hexosaminidase secretion in rabbit lacrimal gland

Cited by 16 publications

References 25 publications

p38 Mitogen-activated protein kinase modulates exocrine secretion in rabbit lacrimal gland

p38 Mitogen-activated protein kinase modulates exocrine secretion in rabbit lacrimal gland

Lacrimal gland primary acinar cell culture: the role of insulin

Traffic of endogenous, transduced, and endocytosed prolactin in rabbit lacrimal acinar cells

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