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

Abstract

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 microM; basal-lateral, BL), and under Cl(-)-free buffer conditions after carbachol stimulation (CCh; 100 microM). 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 beta-hexosaminidase catalytic activity in the AP culture medium in response to 100 microM 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.

Diverse Mediators Modulate the Chloride Ion Fluxes That Drive Lacrimal Fluid Production

“…Reconstituted lacrimal epithelial monolayers generate a negative I SC in response to stimulation with 100 lM CCh; studies with selective inhibitors indicate that it is attributable to a secretory flux of Cl À ions, rather than an absorptive flux of Na þ ions. 39 Unstimulated monolayers consistently generate a small positive I SC ; the ionic basis of this current has not been determined, but current models of 32 As shown in Figure 1, 18-hour exposure to IL-6 at concentrations of 1 and 10 ng/mL and exposure to IL-1b at a concentration of 10 ng/mL decreased CCh-induced I SC by 40%.…”

“…They were maintained for up to 5 days in the Doheny Eye Institute vivaria, a facility fully accredited by the American Association for Laboratory Animal Science, and euthanized as described previously. 39 All protocols conformed to the ARVO Resolution on the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee.…”

“…39 After 2 days, the mediators to be tested were added to both apical and basal chambers, and cells were maintained for 18 to 20 hours more.…”

“…39 Briefly, Ag-AgCl voltage-sensing electrodes and Ag wire current-passing electrodes were connected by agar bridges containing 3 M KCl and interfaced via headstage amplifiers (DM-MC6; Physiologic Instruments, La Mesa, CA) to a microcomputer-controlled voltage/current clamp (VCC-MC6; Physiologic Instruments). Voltage-sensing electrodes were matched to within 1 mV asymmetry and corrected by an offset-removal circuit.…”

“…The introduction of a new ex vivo model, an epithelial monolayer reconstituted from isolated rabbit lacrimal gland acinar cells, 39 has now made it possible to address this question. The magnitude of the secretory Cl À flux the monolayer generates can be measured as the amount of current-referred to as the short-circuit current (I SC )-that must be delivered through an external circuit to eliminate the voltage difference across the monolayer.…”