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) via stimulation of protein kinase C (PKC) in Fischer rat thyroid line (FRTL)-5 thyroid cells. To examine the role of the cAMP pathway in the regulation of PLD, we studied the effects of forskolin (0-100 microM; 30 min) and dibutyryl cAMP (dbcAMP; 0-1 mM; 30 min) on PLD activation. FRTL-5 thyroid cells were labeled mainly in phosphatidylcholine with [3H]myristate followed by incubation with 200 mM ethanol before the addition of agonist. PLD was assessed by the measurement of [3H]phosphatidylethanol. Forskolin (100 nM to 100 microM) and dbcAMP (100 pM to 100 microM) increased PLD activity significantly. Maximal responses to forskolin and dbcAMP exceed the PLD responses produced by 100 microU/ml of TSH. To determine whether the effects of forskolin and dbcAMP on PLD occurred as a consequence of PKC activation, FRTL-5 thyroid cells were preincubated for 10 min with the PKC inhibitors, chelerythrine (1 microM) or calphostin C (1 microM), 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 cAMP agonists. Forskolin (10 microM; 30 min) had no effect on the subcellular distribution of PKC alpha-, epsilon-, or zeta-isoforms, confirming the lack of involvement of PKC. The protein kinase A (PKA) inhibitors, H-89 (10 microM; 30 min) and dideoxyadenosine (5 nM; 10 min) significantly decreased the forskolin- and dbcAMP-mediated PLD activation without any effect on the phorbol ester-mediated PLD response. Following pretreatment with H-89 or dideoxyadenosine, the TSH-mediated PLD response was also significantly reduced. These studies indicate that forskolin and dbcAMP stimulate PLD in FRTL-5 thyroid cells directly via PKA without involvement of PKC. Studies of cells in the presence and absence of ethanol revealed approximately 60% of the phosphatidate plus diacylglycerol produced via TSH occurs via PLD activation. Although TSH-mediated inositol phosphate generation occurred with similar concentrations of TSH that led to PLD activation, 10-fold higher TSH concentrations were required to increase intracellular Ca2+. These results and the lack of a rapid Ca2+ transient following physiological TSH concentrations suggest that alternatives to conventional hydrolysis of phosphatidylinositol 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 these pathways by TSH may be required for optimal physiological activation of PLD.
Ginsberg
J
J
Signal Transduction Laboratories, Department of Medicine, University of Alberta, Edmonton, Canada. Jody.Ginsberg@UAlberta.Ca
Gupta
S
S
Matowe
W C
WC
Kline
L
L
Brindley
D N
DN
D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D003994:Bucladesine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology*; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020404:Glycerophospholipids; D006801:Humans; D009281:Naphthalenes / Q000494:pharmacology; D010617:Phenanthridines / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013961:Thyroid Gland / Q000201:enzymology*; D013972:Thyrotropin / Q000494:pharmacology
D000470:Alkaloids; D053119:Benzophenanthridines; D004075:Diglycerides; D004791:Enzyme Inhibitors; D020404:Glycerophospholipids; D009281:Naphthalenes; D010617:Phenanthridines; D010712:Phosphatidic Acids; C051521:phosphatidylethanol; D005576:Colforsin; D003994:Bucladesine; D013972:Thyrotropin; C016299:chelerythrine; D011493:Protein Kinase C; D010739:Phospholipase D; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium