Abstract
Stimulation of cells with certain agonists often activates both phospholipases C and D. These generate diacylglycerol and phosphatidate, respectively, although the two lipids are also apparently interconvertable through the actions of phosphatidate phosphohydrolase and diacylglycerol kinase. Diacylglycerol activates protein kinase C while one role for phosphatidate is the activation of actin stress fiber formation. Therefore, if the two lipids are interconvertable, it is theoretically possible that an uncontrolled signaling loop could arise. To address this issue structural analysis of diacylglycerol, phosphatidate, and phosphatidylbutanol (formed in the presence of butan-1-ol) from both Swiss 3T3 and porcine aortic endothelial cells was performed. This demonstrated that phospholipase C activation generates primarily polyunsaturated species while phospholipase D activation generates saturated/monounsaturated species. In the endothelial cells, where phospholipase D was activated by lysophosphatidic acid independently of phospholipase C, there was no activation of protein kinase C. Thus we propose that only polyunsaturated diacylglycerols and saturated/monounsaturated phosphatidates function as intracellular messengers and that their interconversion products are inactive.
Highlights
Stimulation of cells with certain agonists often activates both phospholipases C and D
Agonist-stimulated phospholipase D (PLD) activity has been proposed to provide the source of sustained DAG generation in cells leading to the sustained activation of protein kinase C
It is becoming increasingly apparent that the PA product of PLD activation itself functions as an intracellular messenger
Summary
Materials—All solvents were of AnalaR or HPLC grade from Rathburn Chemicals Ltd., Walkerburn, Scotland, United Kingdom. Cell Stimulation—Cells were washed twice with phosphate-buffered saline, preincubated for 10 min in Dulbecco’s modified Eagle’s medium or Ham’s F-12 containing 20 mM Hepes pH 7.4 and 0.1% bovine serum albumin (fraction V) at 37 °C followed by a further 10 min in fresh medium Ϯ butanol before stimulation with 100 nM bombesin (Swiss 3T3 cells) or 10 M sn-1–18:1n-9,2-lysophosphatidic acid (LPA) (PAE cells). Analysis of Protein Kinase C Isozymes—PAE cells were stimulated for 5 min with vehicle, LPA (10 M), or PMA (100 nM), washed 3 times in ice-cold phosphate-buffered saline and harvested in 20 mM Hepes pH 7.4 containing 0.2 mM phenylmethylsulfonyl fluoride and 20 g/ml leupeptin. The basal levels of DAG were 1.4 nmol/107 Swiss 3T3 cells and 13.7 nmol/107 PAE cells
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