Muscarinic regulation of phospholipase D and its role in arachidonic acid release in rat submandibular acinar cells

Abstract

The characteristics of muscarinic cholinergic-induced phospholipase D (PLD) activation, and the involvement of the enzyme in the release of arachidonic acid were examined in rat submandibular acinar cells. Carbachol produced a dose-related activation of PLD to around fivefold control values at 100 microM agonist concentration. This was associated with the appearance of free choline, phosphatidic acid and arachidonic acid, indicating that the PLD substrate was phosphatidylcholine. The response to carbachol was inhibited by 60% by U73122, a blocker of a phospholipase C (PLC) specific to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], suggesting that the cleavage of phosphatidylcholine by PLD was, at least in part, secondary to agonist-coupled hydrolysis of PtdIns(4,5)P2 by PLC. Consistent with this, PLD was also activated to levels comparable to those induced by carbachol, by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the Ca2+ mobilizer, thapsigargin, two agents that respectively mimic the activation of protein kinase C (PKC) by diacylglycerol and the elevation of cytosolic Ca2+ by inositol 1,4,5-triphosphate [Ins(1,4,5)P3] in the phosphoinositide effect. The cell-permeant Ca2+ chelator 1,2-bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA/AM) abolished the thapsigargin-induced activation of PLD and inhibited the responses of PLD to carbachol and TPA by 60%. The PKC inhibitor, Ro-31-8220, also inhibited the activation of PLD by carbacol and TPA to a level of approximately double control values, but had no effect on the thapsigargin-induced elevation of PLD. A role for both the PKC-associated and Ca(2+)-mobilizing arms of the PtdIns(4,5)P2-PLC pathway in PLD regulation is thus suggested. Pretreatment of cells with the phosphatidate phosphohydrolase blocker, propranolol, significantly enhanced the carbachol-induced elevation of phosphatidic acid, but decreased agonist-stimulated production of diacylglycerol and arachidonic acid, indicating that phosphatidlycholine was the likely source of arachidonic acid. We therefore propose that, in submandibular mucous acinar cells, muscarinic activation of the PtdIns(4,5)P2-PLC pathway regulates phosphatidylcholine-specific PLD through both the PKC- and Ca(2+)-mobilizing arms of the phosphoinositide response, and that diacylglycerol, derived from phosphatidylcholine via phosphatidic acid, is a source of free arachidonic acid.

Aluminum fluoride inhibits phospholipase D activation by a GTP‐binding protein‐independent mechanism

“…Fluoride is also known as an inhibitor of phosphatidate phosphohydrolase (PAP) [27], an enzyme that converts the PC hydrolysis product of PLD action, PA, to diacylglycerol (DAG). We previously demonstrated that the PAP inhibitor propranolol (100 μM) lowered DAG and elevated PA in submandibular cells after carbachol stimulation of PLD, raising the possibility of product inhibitory feedback of the enzyme by elevated PA [4]. In the present study, however, we found that inhibition of PAP by propranolol had no effect on PLD activity, suggesting that the AlF 4 − inhibitory effect was independent of its possible action on PAP (results not shown).…”

“…The stimulatory effect of AlF 4 − on PLD was blocked by the aluminum ion chelator deferoxamine (data not shown). From our previous studies on this model, it is assumed that activation of PLD by aluminum fluoride in intact cells results from a stimulatory effect on a heterotrimeric GTP‐binding protein [4,10].…”

“…Submandibular acinar cells from male Sprague‐Dawley rats (200–250 g) were prepared by collagenase dissociation and maintained in short‐term culture in a modified Hanks’ balanced salt solution as described previously [4].…”

“…Cell‐free PLD assays were carried out using phospholipid vesicles (PE/PIP 2 /PC, in a molar ratio of 16:1.4:1) prepared according to Brown et al [16], and containing dipalmitoyl‐[2‐palmitoyl‐9,10‐ 3 H]PC (0.075 μCi per 10 μl lipid vesicle). PLD activity was assayed by quantifying radiolabeled PEth, produced in the transphosphatidylation reaction and isolated by thin layer chromatography [4]. To start the reaction, 10 μl lipid vesicles and 1% ethanol were added to the assay system (50 mM HEPES, 80 mM KCl, 3.0 mM MgCl 2 , 1 mM DTT, 1 mM EGTA, 1 mM CaCl 2 , pH 7.0) which contained 50–100 μg membrane and/or cytosol fraction.…”

Signaling pathways involved in pilocarpine-induced mucin secretion in rat submandibular glands

“…In addition, in the colon, mucin discharge induced by bethanechol requires extracellular calcium and the activation of voltage-dependent calcium channels of Ltype (Barcelo et al, 2001). Finally, the activation of both PKC and NOS during pilocarpine-induced mucin release could be mediated by increases in [Ca 2+ ] I (Chung and Fleming, 1995;Watson et al, 1999). Fig.…”