Involvement of phospholipase D in caerulein-induced phosphatidylcholine hydrolysis in rat pancreatic acini

Abstract

Phosphatidylcholine (PC) metabolism stimulated by caerulein (Cae), a cholecystokinin analogue, was investigated in rat pancreatic acini prelabeled with [3H]choline or [3H]-myristic acid. Both labels were incorporated mostly into PC. An inhibition of choline incorporation into PC was first observed in response to Cae (100 and 500 pM) stimulation, as indicated by reduced [3H]choline incorporation into trichloroacetic acid-precipitable material. Whereas choline incorporation was reduced in PC, Cae (500 pM) significantly increased [3H]choline metabolites release in the incubation medium. Separation of these metabolites by thin-layer chromatography showed that approximately 90% of the labeled products released into the medium was phosphocholine; however, Cae caused significant increases of [3H]choline release after 5, 15, and 30 min. In response to Cae, manoalide, a phospholipase C (PLC) inhibitor, totally prevented phosphocholine release into the medium but did not affect choline release. Staurosporine, a protein kinase C inhibitor, did not influence basal and Cae-induced choline release. In cells prelabeled with [3H]myristic acid, Cae stimulated within 5 min a rapid increase in intracellular [3H]phosphatidic acid (PA) levels in the presence of the PA phosphohydrolase inhibitor, propranolol; this PA production was further increased after 15 and 30 min of stimulation. The time course of [3H]PA formation in the presence of propranolol was similar to that of choline release in the medium. Staurosporine partially blocked PA accumulation stimulated by Cae after 30 min. In contrast, manoalide significantly reduced basal PA accumulation but did not prevent its production in response to Cae. In the presence of ethanol, Cae also significantly stimulated above control values the formation of [3H]phosphatidylethanol. These data indicate that Cae-induced PC hydrolysis in rat pancreatic acini is mediated mostly by phospholipase D (PLD) to produce PA and choline; they suggest a direct action of Cae on PLD activation, an effect independent of PLC activation.

Specificity of Phospholipase D Activation by Cholecystokinin and Phorbol Myristate Acetate but Not by Carbamylcholine and A23187 in Rat Pancreatic Acini

“…Furthermore, this non steroidal sesterterpenoid was reported to be 300-to 1,000-fold less active against mammalian PLAjs than against PLAis isolated from venoms [ 19] and to cause 50% inhibition of CBC-or Cae-stimulated amylase release [7], 300 nM staurosporine was also used to inhibit PK-C. This staurosporine concentration was previously shown to totally inhibit PK-C activity in sonicates of guinea pig pancreatic acini [20], and PMA stimulated amylase release from guinea pig [20] or rat acini [21], At each time period, 1 ml of acini was removed and quickly centrifuged at 10,000 g in a microcentrifuge for 15 s. Aqueous (3H)-cholinc-labeled metabolites were separated by a modi fication of the procedure described by Matozaki and Williams [22] and Vance et al [23] as previously described [7], After TLC separa tion, the plates were developed to the top in a solvent composed of 0.5% NaCl/ethanol/methanol/conccntratcd NH4OH (50:30:20:5. v/ v) [24]. Radioactivity was expressed as the percent of the total radioactivity incorporated into 1 ml of acini.…”

“…PA was sep arated from the chloroform phase in a solvent system containing chloroform/acetone/methanol/acetic acid/water (50:20:15:10:5, v/v) [25] as previously described in details [7]. Radioactivities in PA were expressed as a percent of total radioactivity in the chloroform phase.…”

“…They were then incubated with or without stimuli for the indicated time periods in a medium containing 1% (v/v) ethanol. The plates were developed in a system containing chloroform/methanol/ammonium hydroxide (65:30:3, v/v) [26] as pre viously described in details [7], PEt concentration was normalized to the percent of total labeled lipids.…”

“…PC gives rise to phosphatidic acid and free choline, and the newly formed PA can be transformed into DAG by phosphatidic acid phosphohydrolase [28]. The inhibition of PA phosphohydrolase by propranolol was used to favour the accumulation of PA because we have previously shown that this is a sensitive method to determine PA production in pancreatic acini [7], As shown in figure 4a, in the presence of 200 \xM pro pranolol, 500 pM Cae caused significant increases in PA levels at 5, 15 and 30 min. Under these same conditions, PMA significantly increased levels of PA only after 30 min, whereas 10 pA/ CBC and 1 pA/ of the calcium ionophore A23187 failed to increase PA accumulation.…”

“…There is also some evidence for the involve ment of phospholipase A2 (PLA?) in hormonal signal transduction and enzyme release from pancreatic acinar cells [6], Recently, we have demonstrated [7] that caerulein can activate phospholipase D (PLD) in pancreatic acini by a mechanism which seems independent of PK-C and PLC activation; this finding thus provides an alternate path way in the generation of second messengers derived from membrane phospholipids [8,9]. PLD catalyzes the hydro lysis of the terminal diester bond of phosphatidylcholine (PC) and possibly of other glycerophosphatides with the formation of phosphatidic acid (PA) and choline [8].…”