N-nitro-L-arginine inhibits the inducible heat shock protein 70 kDa through calcium, PKC, and PKA in human intestinal epithelial T84 cells. Am. J. Physiol. XXX (Gastrointestinal and Liver Physiol. xx): Gxx-Gxx, 2001. - It is known that nitric oxide synthase inhibitor N-nitro-L-arginine (LNNA) inhibits heat stress (HS)-induced nitric oxide production and inducible heat shock protein 70 kDa (HSP-70i) in many rodent organs. We used human intestinal epithelial T84 cells to characterize the inhibitory effect of LNNA on the heat stress-induced HSP-70i expression. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured using fluorescent probe Fura-2. PKC and PKA activities in LNNA-treated cells were also determined. HS increased HSP-70i mRNA and protein in T84 cells exposed to 45°C for 10 min and recovered for 6 h. Treatment with LNNA for 1 h prior to HS inhibited the induction of HSP-70i mRNA and protein, with a medium inhibitory concentration (IC₅₀) of 0.0471±0.0007 µM . Since the HS-induced increase in HSP-70i mRNA and protein is calcium-dependent, cytosolic free calcium concentration ([Ca²⁺]_i) was measured after cells were treated with LNNA. LNNA at 100 µM significantly decreased the resting [Ca²⁺]_i from 177±8 nM to 124±5 nM. Likewise, treatment with 1 µM GF-109203X or H89 (inhibitors of PKC and PKA, respectively) for 30 min also significantly decreased [Ca²⁺]_i to 125±6nM and 134±5 nM, respectively, and inhibited the HS-induced increase in HSP-70i. These GF-109203X- or H89-treated cells failed to respond to LNNA by further decreasing [Ca²⁺]_i and HSP-70i. LNNA also effectively blocked the heat shock factor1 (HSF1, a HSP-70i, cytosol-residing, transcription factor) translocation from the cytosol to the nucleus, a process requiring phosphorylation by PKC. These results suggest that LNNA inhibits HSP-70i by reducing [Ca²⁺]_i and decreasing the activity of PKC and PKA, thereby leading to a blockage of HSF1 translocation from the cytosol to the nucleus.