Effect of okadaic acid, a protein phosphatase inhibitor on heat stress-induced HSP72 synthesis and thermotolerance

Heat stress proteins (HSPs), in particular HSP72, seem to play a major role in cell protection against lethal stresses such as hyperthermia or ischemi a. HSP synthesis is negatively regulated by protein phosphatases, which are implicated in dephosphorylation processes. In the present study, we have i nvestigated the effect of okadaic acid (OA, a protein phosphatase inhibitor ) on heat stress-induced HSP72 synthesis and thermotolerance in smooth musc le cells (SMC). SMC were heat stressed (42 degrees C for 20 minutes) in the presence of 250 nM OA (HS+OA cells) or its vehicle (HS+V cells). Control (OA or V) cells w ere not heat stressed. HSP72 mRNA expression was determined 1, 1.5, 3, and 6 hours after heat stress by RT-PCR, and HSP72 synthesis was determined 6, 12, 24, 48, and 72 hours after heat stress by Western blotting. SMC surviva l of lethal hyperthermia (47 degrees C for 90 minutes) was assessed 6, 24, and 48 hours after heat stress by a tetrazolium assay. The maximal expression of HSP72 mRNA was markedly prolonged in HS+OA cells (until 6 hours after heat stress) compared to HS+V cells (1 hour after heat stress). The kinetics of HSP72 synthesis and thermotolerance of SMC were n ot different between HS+OA and HS+V cells. Baseline HSP72 mRNA and protein expression were similar in control Vend OA cells. In conclusion, okadaic acid treatment of SMC potentiated HSP72 mRNA express ion without affecting heat stress-induced HSP72 synthesis and thermotoleran ce.