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Dissertation
Arachidonic acid release by cPLA₂ and ceramide synthesis may be causally related to nitric oxide induced apoptosis in vascular smooth muscle cells
Doctor of Philosophy (Ph.D.), Medical College of Pennsylvania and Hahnemann University
Mar 2002
DOI:
https://doi.org/10.17918/00009785
Abstract
In order to investigate the possible role of arachidonic acid in the apoptosis of smooth muscle, we induced apoptosis in cultured rat aortal smooth muscle cells by treatment with either LTV (Ultraviolet radiation, tumor necrosis factor-[alpha] (TNF-[alpha]) or nitric oxide (NO) donor drugs (sodium nitroprusside (NP), or S-nitroso-N-acetyl penicillamine, SNAP). Apoptosis was detected by either DNA fragmentation analysis of TUNEL analysis. UV radiation and NO were observed to stimulate apoptosis in the cells as well as arachidonic acid release from the cells. They also induced increased levels of cPLA₂ in the cells. These agents stimulated arachidonic acid release somewhat earlier than they stimulated apoptosis in the cells. UV radiation appeared to have a synergistic effect on NP induced cellular apoptosis and arachidonic acid release, when compared with effects of UV radiation, or NP individually. However, when NO levels were determined via Greiss reaction UV radiation was shown to stimulate NO release from either NP or SNAP, thereby greatly increasing the NO concentration in medium. The inhibition of cPLA₂ by arachidonyl trifluoromethyl ketone (AACOCF₃) also led to the inhibition of arachidonic acid release from the cells as well as inhibition of apoptosis. Furthermore, a PLA₂ specific activator peptide, PLAP, showed an increase in arachidonic acid release accompanied by an increase in apoptosis. The cPLA₂ specific inhibitor, AACOCF₃, blocked the PLAP induced effects in a concentration dependent manner. Additional evidence for the involvement of cPLA₂ in VSMC apoptosis was shown by use of the sense and the antisense oligonucleotides. The antisense oligonucleotide abolished cPLA₂ expression in the cells at the highest concentrations used. The effect was accompanied by decrease in arachidonic acid release and apoptosis as stimulated by NP. This observation was not seen with the sense oligonucleotide. These observations provide evidence that arachidonic acid may be involved in apoptosis in VSMCs. Another second messenger lipid investigated was ceramide. Ceramide has been implicated in apoptosis and appears to be involved in NO-induced apoptosis. NP increased the ceramide in smooth muscle cells in a time and NO concentration dependent manner. Addition of exogenous ceramide, in a form of C2-ceramide showed apoptosis, which was not seen with the C2-dihydroceramide analog. The NO-induced ceramide synthesis appears to be made from sphingomyelinase since an inhibitor desipramine decreases ceramide synthesis by NP treated cells. These observations indicated that ceramide is involved in NO induced apoptosis of VSMCs, perhaps as a mediator of apoptosis.
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Details
- Title
- Arachidonic acid release by cPLA₂ and ceramide synthesis may be causally related to nitric oxide induced apoptosis in vascular smooth muscle cells
- Creators
- Cyril M. Pilane
- Contributors
- Joseph T. Nickels (Advisor) - Drexel University, Medical College of Pennsylvania and Hahnemann University (1993-1996, 1998-2002)
- Awarding Institution
- Medical College of Pennsylvania and Hahnemann University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Medical College of Pennsylvania and Hahnemann University; Philadelphia, Pennsylvania
- Number of pages
- xiii, 169 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- School of Medicine (1993-1996, 1998-2002); Medical College of Pennsylvania and Hahnemann University (1993-1996, 1998-2002)
- Other Identifier
- 991021888888404721