Abstract
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.
Publication types
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Calcium / metabolism*
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Cell Nucleus / metabolism
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Chelating Agents / pharmacology
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Colonic Neoplasms
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Cyclic AMP-Dependent Protein Kinases / antagonists & inhibitors
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Cyclic AMP-Dependent Protein Kinases / metabolism
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Cycloheximide / pharmacology
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Cytosol / metabolism
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DNA-Binding Proteins / metabolism
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Dactinomycin / pharmacology
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Egtazic Acid / analogs & derivatives*
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Egtazic Acid / pharmacology
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Enzyme Inhibitors / pharmacology*
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Guanidines / pharmacology
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HSP70 Heat-Shock Proteins / antagonists & inhibitors*
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HSP70 Heat-Shock Proteins / biosynthesis
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HSP70 Heat-Shock Proteins / genetics
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Heat Shock Transcription Factors
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Hot Temperature
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Humans
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Indoles / pharmacology
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Intestinal Mucosa / metabolism*
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Intestines / drug effects
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Isoquinolines / pharmacology
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Maleimides / pharmacology
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Nitric Oxide / metabolism
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Nitric Oxide Synthase / antagonists & inhibitors
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Nitroarginine / pharmacology*
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Protein Kinase C / antagonists & inhibitors
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Protein Kinase C / metabolism
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Protein Kinases / metabolism*
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RNA, Messenger / analysis
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Reverse Transcriptase Polymerase Chain Reaction
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Sulfonamides*
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Transcription Factors
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Tumor Cells, Cultured
Substances
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Chelating Agents
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DNA-Binding Proteins
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Enzyme Inhibitors
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Guanidines
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HSF1 protein, human
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HSP70 Heat-Shock Proteins
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Heat Shock Transcription Factors
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Indoles
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Isoquinolines
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Maleimides
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RNA, Messenger
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Sulfonamides
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Transcription Factors
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Dactinomycin
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Nitroarginine
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Nitric Oxide
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Egtazic Acid
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Cycloheximide
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Nitric Oxide Synthase
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Protein Kinases
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Cyclic AMP-Dependent Protein Kinases
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Protein Kinase C
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1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
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bisindolylmaleimide I
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N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide
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pimagedine
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Calcium