Characterization of β-hexosaminidase secretion in rabbit lacrimal gland

The present study was aimed at validating the use of the lysosomal enzyme β-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 β-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 β-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 β-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 β-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 β-hexosaminidase was easily measured in quantities corresponding to as low as 0.4 μl of tear fluid. Using 4-methylumbelliferyl N-acetyl-β-d-glucosaminide as a substrate for β-hexosaminidase, the Km 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. β-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. β-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.