INHIBITORS OF SER THR PHOSPHATASE-1 AND PHOSPHATASE-2A INDUCE APOPTOSIS IN PITUITARY GH(3) CELLS/

Two structurally different inhibitors of ser/thr phosphatases 1 and 24 , okadaic acid and calyculin A, time- and concentration-dependently st imulated and inhibited cell-specific function (hormone gene expression ) in pituitary GH(3) cells. The negative effect was associated with th e appearance of apoptotic cell death. Nanomolar concentrations of both agents produced the characteristic morphological alterations and a DN A fragmentation ladder. Calyculin A treatment resulted in comparable c hanges with 10fold lower concentrations than okadaic acid. Observation s with derivatives of okadaic acid with no or lower phosphatase inhibi tory potency supported the conclusion that apoptosis induction is rela ted to inhibition of ser/thr phosphatases, presumably types 1 and 2A. Membrane damage as measured by lactate dehydrogenase liberation into m edium was significantly lower in apoptotic vs. necrotic cells. DNA fra gmentation could be reduced by the addition of zinc but not by removal of extracellular calcium with EGTA. Apoptotic changes were reduced by the concomitant activation of protein kinase A by a membrane permeabl e cAMP analogue. Incubation of cells for 4 months in successively incr eased concentrations of okadaic acid resulted in a population that pro liferated at the initially lethal concentration of 30 nM.