Effects of the Protein Phosphatase Inhibitors, Okadaic Acid and Vanadate, on Localization of Occludin in Primary Cultures of Rat Hepatocytes

To elucidate whether protein phosphorylation is associated with the loca-lization of the tight junction protein occludin, we determined the changes of occludin protein expression in primary cultures of rat hepatocytes after treat-ment with the protein phosphatase inhibitors okadaic acid and vanadate. After 2 h of treatment with 1myu M okadaic acid or 5 mM vanadate, occludin immunoreactivity showing continuous lines in non-treated cells changed to a few spots on the plasma membrane. In western blots, broad bands above the occludin protein (65 kD) became conspicuous after treatment with okadaic acid and vanadate. We treated the same samples with alkaline phosphatase to examine whether the broad bands depended on the changes in the phos-phorylation states of occludin protein. The broad bands disappeared and the occludin was observed as a narrow band corresponding to 65 kD. Neither a significant change in the mRNA of occludin nor a change in the immunoreac-tivity of the tight junction associated protein, ZO-1, was observed after treatment with okadaic acid or vanadate. These results suggested that the phosphorylation of occludin is closely associated with localization of the protein in cultured hepatocytes and that protein phosphatase inhibitors affect the loca-lization of occludin but not ZO-1 on the plasma membrane