Characterization of Beta-hexosaminidase Secretion in Rabbit Lacrimal Gland.
From: Department of Chemistry and Biomedical Sciences, University of Kalmar, Smalandsgatan 24, SE-39182 Kalmar, Sweden.
Experimental eye research
- Publish Date: Nov 2006
- ISSN: 0014-4835
- Volume: 83
- Issue: 5
- Pages: 1081-8
- Medium: Print
- Language: English
- Citation (JAMA): Andersson Sofia V, Edman Maria C, Bekmezian Arpi, et al. Characterization of Beta-hexosaminidase Secretion in Rabbit Lacrimal Gland.. Exp. Eye Res. Nov 2006;83:1081-8
Abstract
The present study was aimed at validating the use of the lysosomal enzyme beta-hexosaminidase as a marker of secretory function in cultured rabbit lacrimal gland acinar cells. The secretory response and morphological characteristics of isolated acinar cells cultured in a serum-free medium supplemented with an extracellular matrix extract were monitored over time as part of optimization of our culturing protocol. Secreted beta-hexosaminidase activity was analyzed and compared with that of another lysosomal enzyme, cathepsin B, as well as protein secreted into the media, w or w/o the presence of secretagogues or protein kinase C activators and inhibitors. Lacrimal gland fluid was obtained from pilocarpine stimulated rabbits, and the activities of beta-hexosaminidase and cathepsin B were measured. A membrane fraction and a soluble fraction were obtained from isolated acinar cells and used for kinetic studies of beta-hexosaminidase in comparison with that released from cultured cells, in the lacrimal gland fluid and in serum. Optimal secretory response was obtained when the cells had been in culture for 2-3 days, coinciding with the formation of acinus-like structures. Stimulation of the cultured cells by carbachol or phorbol esters resulted in a more than 3-fold increase of beta-hexosaminidase release over basal, whereas no effect on cathepsin B release could be detected. Treatment with the protein kinase C inhibitor, chelerythrine chloride, significantly decreased the carbachol and phorbol ester-stimulated secretion. Cathepsin B could not be detected in rabbit lacrimal fluid, but beta-hexosaminidase was easily measured in quantities corresponding to as low as 0.4 microl of tear fluid. Using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide as a substrate for beta-hexosaminidase, the K(m) in lacrimal gland fluid (1.22+/-0.15 mM) was not significantly different from that of the membrane-associated fraction, the soluble fraction, rabbit serum or activity secreted from cultured cells. Beta-hexosaminidase is secreted by rabbit lacrimal gland, in vivo, and by acinar cells in primary culture, whereas cathepsin B is not secreted under the conditions described. Beta-hexosaminidase therefore provides a versatile marker for secretion in studies of tear production utilizing the rabbit as a model. Our results also indicate that PKC is an important regulator of rabbit lacrimal gland secretion.
Mesh Headings (Keywords): Alkaloids, Animals, Benzophenanthridines, Carbachol, Cathepsin B, Cells, Cultured, Cholinergic Agonists, Culture Media, Serum-Free, Enzyme Inhibitors, Female, Lacrimal Apparatus, Lysosomes, Phorbol Esters, Protein Kinase C, Rabbits, Signal Transduction, Tears, beta-N-Acetylhexosaminidases
Check for Full Text / PubMed Unique Identifier (PMID): 16839547
This abstract is part of PubMed, a service of the U.S. National Library of Medicine. PubMed includes more than 17 million citations from MEDLINE and other life science journals for biomedical articles. See Copyright and Disclaimers.
Linked medical terms appearing on this page are added by Healia to help readers find more information and are not part of the original PubMed document.
The data herein was last updated on July 8th, 2008 and may not reflect the most current and accurate data available from NLM.