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Eicosanoid/thromboxane A2-independent and -dependent generation of lysoplasmenylethanolamine via phospholipase A2 in collagen-stimulated human platelets.

ABSTRACT: Collagen-induced human platelet stimulation is dependent on the release of arachidonic acid (AA) from membrane phospholipid and the formation of thromboxane A2 (TxA2) for TxA2-induced platelet activation. Since plasmenylethanolamine represents the single major phospholipid reservoir of AA in resting human platelets, we assessed its hydrolysis via phospholipase A2 upon platelet stimulation with low levels of collagen by determining the generation of [3H]lysoplasmenylethanolamine via eicosanoid/TxA2-independent and -dependent processes. Ethanolamine phospholipids in platelets were prelabelled with [3H]ethanolamine before stimulation with either collagen or the TxA2 mimetic U46619, in the presence or absence of BW755C, a dual inhibitor of the cyclooxygenase/lipoxygenase activities, or GR32191B, a TxA2-receptor antagonist. Collagen stimulation promoted a marked generation of [3H]lysoplasmenylethanolamine, which was only moderately decreased when TxA2 synthesis or TxA2 receptors were blocked by BW755C or GR32191B respectively. The moderate rise in [3H]lysoplasmenylethanolamine formation with U46619 as the agonist was only slightly affected by BW755C and blocked by GR32191B. Evidence for eicosanoid/TxA2-independent and -dependent generation of [3H]lysophosphatidylethanolamine was also obtained. A significant quantitative loss of AA from plasmenylethanolamine was also demonstrated in collagen-stimulated platelets. The present findings indicate the activation of plasmenylethanolamine cleavage via phospholipase A2 in collagen-stimulated human platelets, which, to a considerable extent, does not depend on eicosanoid/TxA2 synthesis. This may represent an important source of releasable AA for TxA2 generation and the promotion of further liberation of AA and phospholipid-mediated signalling pathways.

SUBMITTER: Turini ME

PROVIDER: S-EPMC1132223 | biostudies-other | 1993 Feb

REPOSITORIES: biostudies-other

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Project description:Beta-cell mass is regulated by a balance between beta-cell growth and beta-cell death, due to apoptosis. We previously reported that apoptosis of INS-1 insulinoma cells due to thapsigargin-induced ER stress was suppressed by inhibition of the group VIA Ca2+-independent phospholipase A2 (iPLA2beta), associated with an increased level of ceramide generation, and that the effects of ER stress were amplified in INS-1 cells in which iPLA2beta was overexpressed (OE INS-1 cells). These findings suggested that iPLA2beta and ceramides participate in ER stress-induced INS-1 cell apoptosis. Here, we address this possibility and also the source of the ceramides by examining the effects of ER stress in empty vector (V)-transfected and iPLA2beta-OE INS-1 cells using apoptosis assays and immunoblotting, quantitative PCR, and mass spectrometry analyses. ER stress induced expression of ER stress factors GRP78 and CHOP, cleavage of apoptotic factor PARP, and apoptosis in V and OE INS-1 cells. Accumulation of ceramide during ER stress was not associated with changes in mRNA levels of serine palmitoyltransferase (SPT), the rate-limiting enzyme in de novo synthesis of ceramides, but both message and protein levels of neutral sphingomyelinase (NSMase), which hydrolyzes sphingomyelins to generate ceramides, were temporally increased in the INS-1 cells. The increases in the level of NSMase expression in the ER-stressed INS-1 cells were associated with corresponding temporal elevations in ER-associated iPLA2beta protein and catalytic activity. Pretreatment with BEL inactivated iPLA2beta and prevented induction of NSMase message and protein in ER-stressed INS-1 cells. Relative to that in V INS-1 cells, the effects of ER stress were accelerated and/or amplified in the OE INS-1 cells. However, inhibition of iPLA2beta or NSMase (chemically or with siRNA) suppressed induction of NSMase message, ceramide generation, sphingomyelin hydrolysis, and apoptosis in both V and OE INS-1 cells during ER stress. In contrast, inhibition of SPT did not suppress ceramide generation or apoptosis in either V or OE INS-1 cells. These findings indicate that iPLA2beta activation participates in ER stress-induced INS-1 cell apoptosis by promoting ceramide generation via NSMase-catalyzed hydrolysis of sphingomyelins, raising the possibility that this pathway contributes to beta-cell apoptosis due to ER stress.

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Eicosanoid/thromboxane A2-independent and -dependent generation of lysoplasmenylethanolamine via phospholipase A2 in collagen-stimulated human platelets.

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