ACTIVATION OF PHOSPHOLIPASE-D IN FRTL-5 THYROID-CELLS BY FORSKOLIN AND DIBUTYRYL-CYCLIC ADENOSINE-MONOPHOSPHATE

We demonstrated previously that TSH activates phospholipase D (PLD) vi a stimulation of protein kinase C (PKC) in Fischer rat thyroid line (F RTL)-5 thyroid cells. To examine the role of the cAMP pathway in the r egulation of PLD, we studied the effects of forskolin (0-100 mu M; 30 min) and dibutyryl cAMP (dbcAMP; 0-1 mM; 30 min) on PLD activation. FR TL-5 thyroid cells were labeled mainly in phosphatidylcholine with [H- 3]myristate followed by incubation with 200 mM ethanol before the addi tion of agonist. PLD was assessed by the measurement of [H-3]phosphati dylethanol. Forskolin (100 nM to 100 mu M) and dbcAMP (100 pM to 100 m u M) increased PLD activity significantly. Maximal responses to forsko lin and dbcAMP exceed the PLD responses produced by 100 mu U/ml of TSH . To determine whether the effects of forskolin and dbcAMP on PLD occu rred as a consequence of PKC activation, FRTL-5 thyroid cells were pre incubated for 10 min with the PKC inhibitors, chelerythrine (1 mu M) o r calphostin C (1 mu M), or they were pretreated for 24 h with phorbol myristate acetate (100 nM) to down-regulate PKC. Unlike TSH-mediated PLD activation, these treatments had no effect on PLD activation by cA MP agonists. Forskolin (10 mu M; 30 min) had no effect on the subcellu lar distribution of PKC alpha-, epsilon-, or zeta-isoforms, confirming the lack of involvement of PKC. The protein kinase A (PKA) inhibitors , H-89 (10 mu M; 30 min) and dideoxyadenosine (5 nM; 10 min) significa ntly decreased the forskolin- and dbcAMP-mediated PLD activation witho ut any effect on the phorbol eater-mediated PLD response. Following pr etreatment with H-89 or dideoxyadenosine, the TSH-mediated PLD respons e was also significantly reduced. These studies indicate that forskoli n and dbcAMP stimulate PLD in FRTL-5 thyroid cells directly via PKA wi thout involvement of PKC. Studies of cells in the presence and absence of ethanol revealed approximately 60% of the phosphatidate plus diacy lglycerol produced via TSH occurs via PLD activation. Although TSH-med iated inositol phosphate generation occurred with similar concentratio ns of TSH that led to PLD activation, 10-fold higher TSH concentration s were required to increase intracellular Ca2+. These results and the lack of a rapid Ca2+ transient following physiological TSH concentrati ons suggest that alternatives to conventional hydrolysis of phosphatid ylinositol 4,5-bisphosphate may initiate PKC activation. Thus, the two major signal transduction systems in the FRTL-5 thyroid cell (PKA and PKC) appear to converge on PLD activation. Stimulation of both of the se pathways by TSH may be required for optimal physiological activatio n of PLD.