[N.sup.[omega]]-nitro-L-arginine inhibits inducible HSP-70 via [Ca.sup.2+], PKC, and PKA in human intestinal epithelial T84 cells

The nitric oxide (NO) synthase inhibitor [N.sup.[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.sup.2+] concentration ([[[Ca.sup.2+]].sub.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 I[C.sub.50] of 0.0471 [+ or -] 0.0007 [micro]M. Because the HS-induced increase in HSP-70i mRNA and protein is [Ca.sup.2+] dependent, we measured [[[Ca.sup.2+]].sub.i] after treating cells with L-NNA. L-NNA at 100 [micro]M significantly decreased resting [[[Ca.sup.2+]].sub.i]. Likewise, treatment with 1 [micro]M GF-109203X or H-89 (inhibitors of PKC and PKA, respectively) for 30 min also significantly decreased [[[Ca.sup.2+]].sub.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.sup.2+]].sub.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.sup.2+]].sub.i] and decreasing PKC and PKA activity, thereby blocking HSF1 translocation from the cytosol to the nucleus. heat; nitric oxide; heat shock protein; protein kinase C; protein kinase A