Author(s): Huflejt ME, Blum RA, Miller SG, Moore HP, Machen TE
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Abstract We measured stimulant-induced changes of exocytosis that are associated with increases in Cl secretion (i.e., short circuit current, ISC, in microA/cm2) and apical (ap) Cl permeability (PCl) and basolateral (bl) K permeability (PK) (both in cm/s) in T84 monolayers. PCl and PK were measured by permeabilizing the bl or ap membrane with nystatin. PCl was also measured with a fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). A noninvasive and sensitive method (release of 35SO4-labeled glycosaminoglycan [GAG], a fluid-phase marker of Golgi-derived vesicles) was used to measure exocytosis at both ap and bl membranes. At rest, ISC = 3.6, PK = 0.8 x 10(-6), PCl = 2.1 x 10(-6) with SPQ and 2.4 x 10(-6) electrically, and there was constitutive GAG secretion (i.e., exocytosis) to both ap and bl sides (bl > 2 x ap). Carbachol (C) increased: ISC (delta = 18.6), PK (6.5x), PCl (1.8-2.9x), and exocytosis to both ap (2.2-3.5x) and bl (2.0-3.0x) membranes. Forskolin (F) increased ISC (delta = 29), PCl (5.5-11x) and ap exocytosis (1.5-2x), but had no effect on PK or bl exocytosis. Synergistic effects on ISC occurred when C was added to F-treated cells but not vice versa, even though the characteristic effects of F+C on PCl, PK, and/or GAG secretion were identical to those exhibited when stimulants were added individually. Cl secretion results from coordinated activation of channels at ap and bl membranes, and exocytosis may play a role in these events.
This article was published in J Clin Invest
and referenced in Journal of Clinical & Cellular Immunology