Characterization of β-hexosaminidase secretion in rabbit lacrimal gland

Abstract

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 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. 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.