Secretion of alpha-amylase in human parotid gland epithelial cell culture

Abstract

The secretions of the salivary gland system are essential for the maintenance of oral health. The nature of cell-specific secretions of the various glands and their regulation is not completely understood. The objective of this study was to establish epithelial cell cultures from the human parotid gland that exhibit the tissue-specific function of alpha-amylase secretion. A specimen of normal human parotid gland was obtained at surgery and used to obtain primary cultures by the explant/outgrowth procedure. The cultures were maintained in keratinocyte basal medium, supplemented with insulin (5 micrograms/ml), EGF (10 ng/ml), hydrocortisone (0.5 micrograms/ml), bovine pituitary extract (25 micrograms/ml), and antibiotics. The cultures were passaged using 0.125% trypsin to dissociate the cells. Phase contrast and ultrastructural observations showed that the cells were polygonal and exhibited desmosomes. Their cytoplasm contained tonofilament bundles and abundant rough endoplasmic reticulum and Golgi complexes. Immunofluorescence studies showed that all cells were positive for cytokeratins. Immunoblot analysis revealed keratins with molecular weights of 58, 56, 52, 50, 48, 46, and 40 KD, which are characteristic of secretory epithelia. The cells have been passaged 35 times so far, undergoing a cumulative 120-140 population doublings. The serially passaged epithelial cell cultures produced and secreted alpha-amylase, a major component of parotid gland acinar cell secretion. The beta-adrenergic agonist, isoproterenol (ISP), stimulated alpha-amylase secretion, which was accompanied by increased intracellular concentrations of cAMP. ISP-induced stimulation of amylase and cAMP was blocked by the beta-adrenergic antagonist, propranolol. Further, dibutyryl cAMP also enhanced the secretion of amylase. Thus we have established a long-term epithelial cell culture model of human parotid gland epithelial cells that exhibits differentiated function and retains the intact beta-adrenergic receptor system.