Cholinergic activation of phospholipase D in lacrimal gland acini is independent of protein kinase C and calcium

1995 ◽  
Vol 268 (3) ◽  
pp. C713-C720 ◽  
Author(s):  
D. Zoukhri ◽  
D. A. Dartt
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Lacrimal Gland ◽  
Phorbol Esters ◽  
Adrenergic Agonist ◽  
Kinase C ◽  
Cellular Lipids ◽  
Transphosphatidylation Reaction ◽  
Cholinergic Activation

To determine if rat lacrimal gland acini contain phospholipase D (PLD) activity, we took advantage of PLD's unique ability, in the presence of ethanol, to catalyze a transphosphatidylation reaction to produce phosphatidylethanol (PEth). Lacrimal gland acini were labeled for 3 h with [14C]stearic acid, preincubated for 20 min in the presence of 2% ethanol, and incubated for 20 min with or without agonists. Total cellular lipids were then extracted and analyzed by thin-layer chromatography, and the radioactivity was determined by liquid scintillation counting. Carbachol (1 mM), a cholinergic agonist, stimulated the production of both [14C]PEth and [14C]phosphatidic acid ([14C]PA) twofold. This effect was completely blocked by the muscarinic antagonist atropine (10 microM). [14C]PEth accumulation was also stimulated twofold by the active phorbol esters, 4 beta-phorbol 12-myristate 13-acetate and 4 beta-phorbol 12,13-dibutyrate at 1 microM. Ionomycin (1 microM), a Ca2+ ionophore, also stimulated the production of [14C]PEth twofold. In contrast to carbachol, neither phorbol esters nor ionomycin stimulated [14C]PA production. Neither [14C]PEth nor [14C]PA production was altered by epinephrine (1 mM), a nonselective adrenergic agonist, or phenylephrine (0.1 and 1 mM), a specific alpha 1-adrenergic agonist. We concluded that PLD activity, modulated by muscarinic receptors, protein kinase C, and Ca2+, but not by adrenergic receptors, is present in rat lacrimal gland acini. We also concluded that cholinergic activation of PLD appears to be independent of PKC and Ca2+.

Receptor- and phorbol ester-mediated phospholipase D activation in rat parotid involves two different pathways

1994 ◽  
Vol 266 (3) ◽  
pp. C692-C699 ◽  
Author(s):  
I. Guillemain ◽  
B. Rossignol
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Phorbol Esters ◽  
Calcium Ionophore ◽  
Transferase Activity ◽  
Base Exchange ◽  
Calcium Dependent ◽  
Kinase C ◽  
Rat Parotid

We have investigated phospholipase D (PLD) activation in rat parotid acini prelabeled with [14C]stearic acid. In the presence of 2% ethanol, muscarinic and alpha-adrenergic agonists stimulated the formation of [14C]phosphatidylethanol as a result of a PLD activity. The calcium ionophore, ionomycin, and the phorbol esters, 4 beta-phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu), also stimulated phosphatidylethanol accumulation, but 1-oleyl-2-acetyl-sn-glycerol (OAG), a permeant analogue of diacylglycerol did not. Chelerythrine and staurosporine, two inhibitors of protein kinase C, failed to affect any response. These results suggest that protein kinase C was not involved in the regulation of PLD activity. A difference between PLD regulation by PMA and receptor-mediated agonists was observed with regard to the extracellular calcium requirement. Our results strongly suggest that PLD activation in parotid acini involved different pathways: a calcium-dependent pathway activated by receptor-mediated agonists and a calcium-independent pathway activated by phorbol esters. Moreover, we observed that PLD activation did not result in any change in phosphatidic acid level. We propose that the phosphatidyl transferase activity of PLD reflected a metabolic pathway which may allow a base-exchange reaction in parotid gland.

scholarly journals Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1.

1991 ◽  
Vol 2 (11) ◽  
pp. 897-903 ◽  
Author(s):  
J K Pai ◽  
E A Dobek ◽  
W R Bishop
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Phorbol Esters ◽  
Thymidine Incorporation ◽  
Maximal Effect ◽  
Kinase C ◽  
Potent Vasoconstrictor ◽  
Dose Dependent ◽  
Protein Kinase C Beta

Endothelins (ETs) are a family of extremely potent vasoconstrictor peptides. In addition, ET-1 acts as a potent mitogen and activates phospholipase C in smooth muscle cells and fibroblasts. We examined the effects of ET-1 on phosphatidylcholine (PC) metabolism and thymidine incorporation in control Rat-6 fibroblasts and in cells that overexpress protein kinase C beta 1 (PKC). PC pools were labeled with [3H]myristic acid, and formation of phosphatidylethanol (PEt), an unambiguous marker of phospholipase D (PLD) activation, was monitored. ET-1 stimulated much greater PEt formation in the PKC overexpressing cells. ET-1 action was dose-dependent with a half-maximal effect at 1.0 x 10(-9) M. With increasing ethanol concentrations, [3H]PEt formation increased at the expense of [3H]phosphatidic acid (PA). Propranolol, an inhibitor of PA phosphohydrolase, increased [3H]PA accumulation and decreased [3H]diacylglycerol (DAG) formation. These data are consistent with the formation of [3H]DAG from PC by the sequential action of PLD and PA phosphohydrolase. Phorbol esters are known to stimulate thymidine incorporation and PLD activity to a greater extent in PKC overexpressing cells than in control cells. ET-1 also stimulates thymidine incorporation to a greater extent in the PKC overexpressing cells. The effect of ET-1 on thymidine incorporation into DNA in the overexpressing cells was also dose-dependent with a half-maximal effect at 0.3 x 10(-9) M. Enhanced PLD activity induced by ET-1 in the overexpressing cells may contribute to the mitogenic response, especially in light of a possible role of the PLD product, PA, in regulation of cell growth.

scholarly journals Differential regulation of phospholipase D and phospholipase A2 by protein kinase C in P388D1 macrophages

1997 ◽  
Vol 321 (3) ◽  
pp. 805-810 ◽  
Author(s):  
Jesús BALSINDE ◽  
María A. BALBOA ◽  
Paul A. INSEL ◽  
Edward A. DENNIS
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase A2 ◽  
Phospholipase D ◽  
Phorbol Esters ◽  
Platelet Activating Factor ◽  
Lipid Mediator ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Pkc Activation

Activation of P388D1 macrophages by phorbol myristate acetate (PMA) resulted in the translocation of the protein kinase C (PKC) isoforms α, Δ, and ε from the cytosol to membranes. Furthermore, PMA activated phospholipase D (PLD) in these cells, and potentiated the effect of the inflammatory lipid mediator platelet-activating factor (PAF) on PLD activation. PAF also activated phospholipase A2 (PLA2) and enhanced arachidonic acid (AA) release in P388D1 macrophages, and bacterial lipopolysaccharide (LPS) increased the responsiveness of these cells to PAF. In contrast with PLD, PLA2 activation in P388D1 macrophages was found to take place independently of PKC. This was supported by the following evidence: (i) PMA neither induced AA release nor enhanced the PAF response; (ii) inclusion of PMA along with LPS during priming did not have any effect on PAF-stimulated AA release; (iii) down-regulation of PMA-activatable PKC isoforms by chronic treatment with the phorbol ester had no effect on the PAF response; and (iv) the PKC inhibitor staurosporine did not alter the PAF-induced AA release. The present study provides an example of cells in which the direct activation of PKC by phorbol esters does not lead to a primed and/or enhanced AA release. As a unique example in which PKC activation is neither necessary nor sufficient for AA release to occur, this now allows study of the separate and distinct roles for PLD and PLA2 in signal-transduction processes. This has hitherto been difficult to achieve because of the lack of specific inhibitors of these two phospholipases.

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