Thyrotropin-releasing hormone increases phospholipase D activity through stimulation of protein kinase C in GH3 cells.

Activation of phospholipase D was investigated after treatment of GH3 cells with thyrotropin-releasing hormone. Thyrotropin-releasing hormone treatment resulted in both time- and dose-dependent increases of phospholipase D activity, translocation of protein kinase C-alpha and -beta I isozymes from cytosol to membrane within 30 min, and approx 43-fold increase of phosphatidylinositol-specific phospholipase C activity. Intracellular calcium concentration was rapidly increased and diacyglycerol level remained high up to 3 h after the treatment. Pretreatment of the cells with U73122, a potent inhibitor of phosphatidylinositol-specific phospholipase C, inhibited thyrotropin-releasing hormone-induced phospholipase D activation. Protein kinase C activity was down-regulated by pretreatment of the GH3 cells with either protein kinase C inhibitors (RO320432, GF109203X) or preincubation of the cells with phorbol myristrate acetate (500 nM) for 24 h. This treatment largely abolished the thyrotropin-releasing hormone-induced activation of phospholipase D, thus further confirming the involvement of protein kinase C in the activation. These results suggest that thyrotropin-releasing hormone-induced phospholipase D activation may be due to phosphatidylinositol-specific phospholipase C, and activation of protein kinase C isozymes is responsible for this stimulation.