Characterization of beta-hexosaminidase secretion in rabbit lacrimal gland.
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.
Andersson
Sofia V
SV
Department of Chemistry and Biomedical Sciences, University of Kalmar, Smalandsgatan 24, SE-39182 Kalmar, Sweden.
Edman
Maria C
MC
Bekmezian
Arpi
A
Holmberg
Jens
J
Mircheff
Austin K
AK
Gierow
J Peter
JP
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D053119:Benzophenanthridines / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002401:Cathepsin B / Q000378:metabolism; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D007765:Lacrimal Apparatus / Q000187:drug effects / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011817:Rabbits; D015398:Signal Transduction; D013666:Tears / Q000187:drug effects / Q000201:enzymology; D001619:beta-N-Acetylhexosaminidases / Q000032:analysis / Q000378:metabolism*
D000470:Alkaloids; D053119:Benzophenanthridines; D018679:Cholinergic Agonists; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D010703:Phorbol Esters; D002217:Carbachol; C016299:chelerythrine; D011493:Protein Kinase C; D001619:beta-N-Acetylhexosaminidases; D002401:Cathepsin B