Transepithelial bioelectrical properties of rabbit acinar cell monolayers on polyester membrane scaffolds.
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.
Selvam
Shivaram
S
Mork Family Department of Chemical Engineering and Materials Science, Viterbi School of Engineering, University of Southern California, Los Angeles, CA, USA.
Thomas
Padmaja B
PB
Gukasyan
Hovhannes J
HJ
Yu
Alan S
AS
Stevenson
Douglas
D
Trousdale
Melvin D
MD
Mircheff
Austin K
AK
Schechter
Joel E
JE
Smith
Ronald E
RE
Yiu
Samuel C
SC
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods; D002712:Chlorides / Q000378:metabolism; D017097:Electric Impedance; D004594:Electrophysiology; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D005260:Female; D005455:Fluorescent Antibody Technique; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D007765:Lacrimal Apparatus / Q000166:cytology / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008567:Membranes, Artificial; D046529:Microscopy, Electron, Transmission; D008954:Models, Biological; D062793:Occludin; D010042:Ouabain / Q000494:pharmacology; D011091:Polyesters; D011817:Rabbits; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D019108:Tight Junctions / Q000378:metabolism / Q000648:ultrastructure; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D002712:Chlorides; D008565:Membrane Proteins; D008567:Membranes, Artificial; D062793:Occludin; D011091:Polyesters; D002034:Bumetanide; D010042:Ouabain; D000584:Amiloride; D002217:Carbachol; D001619:beta-N-Acetylhexosaminidases; D000254:Sodium-Potassium-Exchanging ATPase