Regulated Cl transport, K and Cl permeability, and exocytosis in T84 cells.

Abstract

We measured stimulant-induced changes of exocytosis that are associated with increases in Cl secretion (i.e., short circuit current, Isc, in gA/cm2) and apical (ap) Cl permeability (Pa) and basolateral (bl) K permeability (PK) (both in cm/s) in T84 monolayers. Pc0 and PK were measured by permeabilizing the bl or ap membrane with nystatin. Pc0 was also measured with a fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). A noninvasive and sensitive method (release of 3`So4-labeled glycosaminoglycan IGAGI, 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, Pc0 = 2.1 x 10-' 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 (A = 18.6), PK (6.5X), Pc, ( 1.8-2.9x ), and exocytosis to both ap (2.2-3.5x) and bl (2.0-3.0x) membranes. Forskolin (F) increased ISC (A = 29), PC0 (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 Pc,, PKI 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. (J. Clin.

Proton leak and CFTR in regulation of Golgi pH in respiratory epithelial cells

“…2-4), and bafilomycin-induced alkalinization data were obtained from the literature (Table 2). These P H ϩ values are large compared with typical permeability values for Na ϩ , K ϩ , and Cl Ϫ : 10 Ϫ12 cm/s for a membrane without channels (37) and up to 10 Ϫ5 cm/s for a membrane with channels (22,24). However, our estimates of P H ϩ compare favorably with P H ϩ in lipid bilayers and liposomes: 10 Ϫ7 -10 Ϫ2 cm/s (8,21,37,40).…”

A Natural Plant-Derived Dihydroisosteviol Prevents Cholera Toxin-Induced Intestinal Fluid Secretion

“…If the basolateral membrane is permeabilized by amphotericin B in the presence of a chloride gradient, any current stimulated by forskolin would reflect chloride transport through the cAMPactivated chloride channel in the apical membrane. Dihydroisosteviol effectively inhibited cAMP-activated Cl Ϫ current under this condition in a dose-dependent manner with an apparent IC 50 value of 5 M. In T84 cells, CFTR provided the principal route for chloride exit across the apical membrane in the process of cAMP-activated chloride secretion (Huflejt et al, 1994), and thus these findings indicate that dihydroisosteviol may act at the apical CFTR-Cl Ϫ channel to inhibit cAMP-activated chloride secretion. This finding is supported by the results showing that dihydroisosteviol inhibited chloride secretion induced by a cell-permeable cAMP analog (8cpt-cAMP).…”

Subcellular distribution and targeting of the intracellular chloride channel p64.

“…* , " ; < > , . (Huflejt et al, 1994). We asked whether p64 vesicles belong to a secretory pathway by monitoring the distribution of p64 following treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA).…”