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METHODS IN CELL PHYSIOLOGY

Transepithelial bioelectrical properties of rabbit acinar cell monolayers on polyester membrane scaffolds

¹Mork Family Department of Chemical Engineering and Materials Science, Viterbi School of Engineering, University of Southern California, Los Angeles; ²Ocular Surface Center, Doheny Eye Institute, Los Angeles; ³La Jolla Laboratories, Pfizer Inc., San Diego; Departments of ⁴Medicine, ⁵Ophthalmology, ⁶Physiology and Biophysics, and ⁷Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, California

Submitted 16 May 2007 ; accepted in final form 12 August 2007

In our quest to develop a tissue-engineered tear secretory system, we have tried to demonstrate active transepithelial ion fluxes across rabbit lacrimal acinar cell monolayers on polyester membrane scaffolds to evaluate the bioelectrical properties of the cultured cells. Purified lacrimal gland acinar cells were seeded onto polyester membrane inserts and cultured to confluency. Morphological properties of the cell monolayers were evaluated by transmission electron microscopy and immunofluorescence staining for Na⁺,K⁺-ATPase and the tight junction-associated protein occludin. Sections revealed cell monolayers with well-maintained epithelial cell polarity, i.e., presence of apical (AP) secretory granules, microvilli, and junctional complexes. Na⁺,K⁺-ATPase was localized on both the basal-lateral and apical plasma membranes. The presence of tight cell junctions was demonstrated by a positive circumferential stain for occludin. Bioelectrical properties of the cell monolayers were studied in Ussing chambers under short-circuit conditions. Active ion fluxes were evaluated by inhibiting the short-circuit current (I_sc) with a Na⁺,K⁺-ATPase inhibitor, ouabain (100 µM; basal-lateral, BL), and under Cl^–-free buffer conditions after carbachol stimulation (CCh; 100 µM). The directional apical secretion of Cl^– was demonstrated through pharmacological analysis, using amiloride (1 mM; BL) and bumetanide (0.1 mM; BL), respectively. Regulated protein secretion was evaluated by measuring the -hexosaminidase catalytic activity in the AP culture medium in response to 100 µM basal CCh. In summary, rabbit lacrimal acinar cell monolayers generate a Cl^–-dependent, ouabain-sensitive AP BL I_sc in response to CCh, consistent with current models for Na⁺-dependent Cl^– secretion.

lacrimal gland; short-circuit current; epithelial ion channels; Na⁺/H⁺ exchangers; Na⁺-K⁺-2Cl^– symporters

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