phosphatidylethanol and chelerythrine

Receptor-mediated activation of phosphatidylcholine phosphatidohydrolase or phospholipase D (PLD) was studied in Chinese hamster ovary (CHO) cells expressing the cholecystokinin-A (CCK-A) receptor. Cells were labelled with [3H]myristic acid for 24 h and PLD-catalysed [3H]phosphatidylethanol formation was measured in the presence of 1% (v/v) ethanol. Cholecystokinin-(26-33)-peptide amide (CCK8) increased PLD activity both time- and dose-dependently. Maximal activation of protein kinase C (PKC) with 1 microM PMA or sustained elevation of the cytosolic free Ca2+ concentration ([Ca2+]i) with 1 microM thapsigargin increased PLD activity to 50% and 70% of the maximal value obtained with CCK8 respectively. The stimulatory effects of CCK8, PMA and thapsigargin were abolished in cells in which PKC was downregulated or inhibited by chelerythrine. PMA/Ca2+-stimulated PLD activity was absent in a homogenate of PKC-downregulated cells but could be restored upon addition of purified rat brain PKC. CCK8-induced PLD activation was inhibited by 90% in the absence of external Ca2+, demonstrating that receptor-mediated activation of PKC in itself does not significantly add to PLD activation but requires a sustained increase in [Ca2+]i. Taken together, the results presented demonstrate that, in CHO-CCK-A cells, receptor-mediated PLD activation is completely dependent on PKC, but that the extent to which PLD becomes activated depends largely, if not entirely, on the magnitude and duration of the agonist-induced increase in [Ca2+]i.

Topics: Alkaloids; Animals; Benzophenanthridines; Brain; Calcium; CHO Cells; Cricetinae; Cytosol; Down-Regulation; Enzyme Activation; Glycerophospholipids; Phenanthridines; Phospholipase D; Protein Kinase C; Protein Kinase Inhibitors; Rats; Receptor, Cholecystokinin A; Receptors, Cholecystokinin; Recombinant Proteins; Sincalide; Tetradecanoylphorbol Acetate; Thapsigargin

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

Topics: Alkaloids; Animals; Benzophenanthridines; Bucladesine; Calcium; Cell Line; Colforsin; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Glycerophospholipids; Humans; Naphthalenes; Phenanthridines; Phosphatidic Acids; Phospholipase D; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate; Thyroid Gland; Thyrotropin

We have previously demonstrated that muscarinic and alpha-adrenergic receptors regulated a phospholipase D (PLD) activity in parotid glands. Since phorbol 12-myristate, 13-acetate (PMA) induced production of phosphatidylethanol (PEt), a stable metabolite widely accepted as marker of PLD activation, we have investigated the role of protein kinase C (PKC) in PLD stimulation in parotid acini. We tested PKC inhibitors on PEt formation elicited by PMA, by muscarinic and adrenergic agents. Staurosporine and chelerythrine, which act on the catalytic domain of PKC, did not allow the attribution of a role for PKC in PLD activation. Indeed, staurosporine did not affect PMA-mediated PLD activity and chelerythrine showed an important non-specific effect, independent of PKC inhibition. On the other hand, calphostin C, which acts on the regulatory domain of PKC, affected PMA- and receptor-mediated PLD stimulation. We attributed this effect to PKC inhibition and we suggested PKC involvement in PLD regulation in parotid gland. Since only PKC inhibitor acting on the regulatory part of the enzyme affected PLD activity, we also suggested that PKC could be involved in PLD activation through a pathway independent of the phosphorylation mechanism.

Topics: Alkaloids; Animals; Benzophenanthridines; Enzyme Activation; Glycerophospholipids; Male; Naphthalenes; Parotid Gland; Phenanthridines; Phosphatidic Acids; Phospholipase D; Polycyclic Compounds; Protein Kinase C; Rats; Rats, Sprague-Dawley; Staurosporine; Tetradecanoylphorbol Acetate