Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective transepithelial Cl- transport. The regulation of CF gene expression is not fully understood. We report that interferon-gamma (IFN-gamma), but not IFN-alpha or -beta, downregulates CFTR mRNA levels in two colon-derived epithelial cell lines, HT-29 and T84, in a time- and concentration (from 0.1 IU/ml)-dependent manner. IFN-gamma has no effect on the transcription rate of the CFTR gene but reduces CFTR mRNA half-life, indicating that it exerts a posttranscriptional regulation of CFTR expression, at least partly, through destabilization of the transcripts. Cells treated with IFN-gamma contain subnormal amounts of 165-kDa CFTR protein. Assays of adenosine 3',5'-cyclic monophosphate-stimulated 36Cl- efflux and whole cell currents show that CFTR function is diminished in IFN-gamma-treated cells. IFN-gamma and tumor necrosis factor-alpha synergistically reduce CFTR gene expression. Our results suggest that production of these cytokines in response to bacterial infections and inflammatory disorders may alter transmembrane Cl- transport.
The elementary K+ conductance activated by the cAMP or the Ca2+ second messenger pathways was investigated in the model salt-secreting epithelium, the human T84 cell line. Under Cl(-)-free conditions, an inwardly rectifying whole-cell K+ current was evoked by either forskolin 10 (mumol/l) or acetylcholine 1 (mumol/l) and blocked by extracellular charybdotoxin 10 (nmol/l). In the cell-attached mode, both secretory agonists induced the opening of a channel showing inward rectification with a unitary chord conductance of 36.8 +/- 2.5 pS (n = 26) for inward currents. In inside-out patches, a 35-pS inwardly rectifying K+ channel that corresponded to the channel recorded in the cell-attached configuration was recorded in the presence of 0.3 mumol/l free Ca2+ at the inner side of the membrane. This channel was blocked by Ba2+ (5 mumol/l) and by charybdotoxin (50 nmol/l). Its open probability was enhanced by intracellular Ca2+ with and EC50 of 0.25 mumol/l and strongly reduced by intracellular MgATP with an IC50 of 600 mumol/l. In the continuous presence of ATP, the channel activity was consistently increased by 125 kU/l catalytic subunit of cAMP-dependent protein kinase. In the cystic fibrosis pancreatic duct cell line CFPAC-1, a K+ channel was also recorded, with similar characteristics and regulation as the 35-pS channel in T84 cells. We conclude that an ATP-sensitive K+ channel regulated by intracellular Ca2+ and phosphorylation supports the main K+ current activated by secretory agonists in normal cystic fibrosis cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)
1. Whole-cell currents were investigated in the model salt-secreting epithelium, human T84 cell line, by means of the perforated patch-clamp technique. In the control extracellular medium containing Cl-, depolarizing voltage ramps evoked current responses which peaked at 5A43 + 0-81 pA pF-' at +60 mV and had a reversal potential (Erev) of -38-4 + 2-5 mV (n = 23).2. Activation of the cAMP pathway with forskolin increased the current at +60 mV from (n =6) and from 6-36 to 34-13 pA pF' (n = 4), respectively. With both agonists, Erev was shifted either towards the reversal potential for potassium, EK, or towards the reversal potential for chloride, Ecl, depending on the cell.4. In the absence of chloride ions (gluconate substituted), stimulation of the Ca2+ pathway activated a time-independent outward current of large amplitude. This current exhibited inward rectification at positive voltages, reverted at -89-5 + 0-2 mV and was markedly reduced by charybdotoxin (10 nM), a specific blocker of Ca2+-activated K+ channels. When a voltage step protocol was used, increased [Ca2+]i also activated an outward current at potentials more positive than -40 mV which slowly relaxed during depolarizing steps. 5. The activation of both (i) a time-independent inwardly rectifying charybdotoxinsensitive K+ current, and (ii) a time-dependent slowly inactivating current was also produced by cAMP stimulation. 6. We concluded that (i) in the T84 epithelial cells, both Cl-and K+ currents are concomitantly increased by secretagogue stimulation, and (ii) two different types of K+ conductances are activated by either the cAMP or the intracellular Ca21 secreting pathways.
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