N(omega)-nitro-L-arginine inhibits inducible HSP-70 via Ca(2+), PKC, and PKA in human intestinal epithelial T84 cells.
The nitric oxide (NO) synthase inhibitor N(omega)-nitro-L-arginine (L-NNA) inhibits heat stress (HS)-induced NO production and the inducible 70-kDa heat shock protein (HSP-70i) in many rodent organs. We used human intestinal epithelial T84 cells to characterize the inhibitory effect of L-NNA on HS-induced HSP-70i expression. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using fura-2, and protein kinase C (PKC), and PKA activities were determined. HS increased HSP-70i mRNA and protein in T84 cells exposed to 45 degrees C for 10 min and allowed to recover for 6 h. L-NNA treatment for 1 h before HS inhibited the induction of HSP-70i mRNA and protein, with an IC(50) of 0.0471 +/- 0.0007 microM. Because the HS-induced increase in HSP-70i mRNA and protein is Ca(2+) dependent, we measured [Ca(2+)](i) after treating cells with L-NNA. L-NNA at 100 microM significantly decreased resting [Ca(2+)](i). Likewise, treatment with 1 microM GF-109203X or H-89 (inhibitors of PKC and PKA, respectively) for 30 min also significantly decreased [Ca(2+)](i) and inhibited HS-induced increase in HSP-70i. GF-109203X- or H-89-treated cells failed to respond to L-NNA by further decreasing [Ca(2+)](i) and HSP-70i. L-NNA effectively blocked heat shock factor-1 (HSF1) translocation from the cytosol to the nucleus, a process requiring PKC phosphorylation. These results suggest that L-NNA inhibits HSP-70i by reducing [Ca(2+)](i) and decreasing PKC and PKA activity, thereby blocking HSF1 translocation from the cytosol to the nucleus.
Kiang
Juliann G
JG
Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring 20910-7500, USA. Juliann.Kiang@na.amedd.army.mil
Kiang
Sharon C
SC
Juang
Yuang-Taung
YT
Tsokos
George C
GC
D002118:Calcium / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D003110:Colonic Neoplasms; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006146:Guanidines / Q000494:pharmacology; D018840:HSP70 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D000076249:Heat Shock Transcription Factors; D006358:Hot Temperature; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000187:drug effects; D007546:Isoquinolines / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D019335:Nitroarginine / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013449:Sulfonamides; D014157:Transcription Factors; D014407:Tumor Cells, Cultured
D002614:Chelating Agents; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D006146:Guanidines; C000619706:HSF1 protein, human; D018840:HSP70 Heat-Shock Proteins; D000076249:Heat Shock Transcription Factors; D007211:Indoles; D007546:Isoquinolines; D008301:Maleimides; D012333:RNA, Messenger; D013449:Sulfonamides; D014157:Transcription Factors; D003609:Dactinomycin; D019335:Nitroarginine; D009569:Nitric Oxide; D004533:Egtazic Acid; D003513:Cycloheximide; D019001:Nitric Oxide Synthase; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; C070515:bisindolylmaleimide I; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; C004479:pimagedine; D002118:Calcium