Aim:Interferon-gamma (IFN) is well known to chronically down-regulate intestinal epithelial ion transport and barrier function, largely through specific decreases in protein expression such as the alpha subunit of the Na pump, NalK/2CI co-transporter, occ1udin, and ZO proteins.Although the cellular events mediating these effects were not known, we observed that IFN causes an acute inhibition of Na,K-ATPase (Na pump) activity.Therefore, we tested the hypothesis that this event, which leads to increases intracellular Na (Na.) and cell volume, mediates the chronic IFN effects on epithelial function.Methods: I) Na pump activity, Na., and cell volume were measured in T84 colon cells incubated with lOOOIU IFN for 6 or 24 hours.2) Expression of transport-and barrier-function associated proteins were determined by Western blot after 6 or 24 hours in control cells and cells subjected to low extracellular Na (20mM), hypotonic stress, ouabain, and monensin.Results: Treatment of cells with IFN, ouabain, and monensin caused significant increases in Nai and cell volume and nearly identical downregulatory effects by 24 h on barrier-and transport-related proteins.These effects were blocked by incubating cells in 20mM Na-containing media, which prevented IFN-induced effects on Na, and cell volume.These results strongly implicated the downregulation of Na pump activity as an important initiating signal.To determine the role of increased cell volume in mediating the IFN and Ouabain effects, cell were subjected to chronic hypotonic stress.Hypotonic-induced cell swelling had similar downregulatory effects as these agents, but notably did not affect the expression of the alpha! subunit of the Na pump.Thus, increased Na, resulting from chronic IFN or Ouabain treatment may be a primary determinant of the downregulated alpha] subunit of the Na pump.Conclusion:The selective downregulation of intestinal epithelial transport-and barrier-function related proteins by IFN is secondary to an initial inhibition of Na,K-ATPase activity.This event results in increased Na, and cell volume which are distinct signaling events that ultimately downregulate specific intestinal epithelial functions.These effects may be important for phenotypic transformation or for reducing metabolic requirements during periods of sustained inflammation-associated stress.

ABSTRACT The synthesis of the vasoconstrictor peptide endothelin-2 (ET-2) is dependent on hydrolysis of the biologically inactive intermediate big ET-2 by an endothelin-converting enzyme (ECE). Here, mechanisms inducing ET-2 synthesis have been investigated using the human renal adenocarcinoma cell line (ACHN). Synthesis of ET-2 by ACHN cells was inhibited by phosphoramidon (IC(50( congruent with11 microM). To determine whether ET-2 synthesis occurs in parallel with the metallopeptidase ECE-1, a putative processing peptidase for big ET-2, changes in the levels of their mRNAs were compared by semi-quantitative RT-PCR under conditions causing the upregulation of ET-2 synthesis. Tumour necrosis factor-alpha (TNFalpha), forskolin and a cell-permeable cAMP analogue (dibutyryl cAMP) caused concentration-dependent increases in ET-2 synthesis. Combination of forskolin or dibutyryl cAMP with TNFalpha produced a significantly greater increase in ET-2 production than these agents alone, indicating that adenylate cyclase and TNFalpha induce ET-2 synthesis by separate signalling pathways. Studies using receptor selective TNFalpha mutants, (125(I-TNFalpha binding and TNF receptor mRNA showed that type-1 TNF receptors mediate the ET-2 response to TNFalpha. PreproET-2 mRNA levels were increased by TNFalpha at 1 h and 2 h, but returned to control levels at 4 h. Treatment with forskolin significantly increased preproET-2 mRNA levels after 1 h and 4 h. ACHN cells expressed ECE-1b and ECE-1c, but not the ECE-1a isoform of this peptidase. RT-PCR for the combined isoforms ECE-1b/c/d showed TNFalpha to increase mRNA levels at 2 h and 4 h. Forskolin had no effect on ECE-1b/c/d mRNA levels. Thus, expression of ET-2 and ECE-1b/c/d mRNAs in ACHN cells do not display the co-ordinated regulation observed with typical peptide prohormone processing enzymes and their substrates.

The effects on the expression and secretion of ET-2 of forskolin and tumor necrosis factor-alpha (TNF-alpha) were investigated using the human renal adenocarcinoma (ACHN) cell line. For comparison, changes in endothelin-converting enzyme-1 b (ECE-1 b) [corrected] mRNA were also determined. Treatment for 4 with TNF-alpha (3 ng/ml), forskolin (30 microM), or the combination caused significant increases in ET-2 release, TNF-alpha alone or in combination with forskolin increased ET-2 mRNA levels at 1 h and 2 h. After 4 h the expression of ET-2 mRNA was comparable to control levels. In contrast to ET-2, ECE-1 b [corrected] mRNA levels were increased by TNF-alpha only at 4 h. Forskolin increased expression of ET-2 mRNA at 1 and 4 but had no significant effect on expression of ECE-beta. These data suggest that expression of ET-2 and ECE-1 b [corrected] mRNA is regulated differently in ACHN cells.