Abstract
In human basophils, degranulation stimulated by receptor activation or Ca++ ionophores is accompanied by an increase in free arachidonic acid (AA) as determined by gas chromatography negative ion chemical ionization mass spectrometry. Previous studies suggested that there was more than one pool of AA generated during stimulation and indirectly suggested that the leukotriene (LTC4) generated in these reactions was dependent on only one of these pools of AA. Our studies further examined these issues. Preliminary studies demonstrated discordance in the generation of free AA and LTC4 release. Treatment of basophils with triacsin C, a reacylation inhibitor, led to a marked increase in N-formyl-l-methionyl-l-leucyl-l-phenylalanine-(fMLP) stimulated free AA generation with no effect on LTC4release. Similarly, incubation of basophils with recombinant human secretory phospholipase A2 (sPLA2), before and during fMLP stimulation, led to the generation of extremely high levels of free AA with no effect on LTC4 release. Pretreatment of basophils with anti-14 kDa phospholipase A2 monoclonal antibody (mAb 3F10) inhibited fMLP-induced synthesis of LTC4 but did not attenuate the mass of AA measured nor histamine release. Treating human basophils with zileuton (an inhibitor of 5-lipoxygenase) inhibited the stimulated synthesis of LTC4 and in combination with triacsin C increased the amount of observable AA by an amount approximately equal to the loss in LTC4 mass. Monoclonal antibody 3F10 blocked only the enhanced AA production caused by the combination of zileuton and triacsin C. Monoclonal antibody 3F10 did not inhibit the increases in free AA produced by pretreatment with triacsin C alone. These findings were supported by experiments using another relatively specific inhibitor of sPLA2, SB 203347. In all respects, SB 203347 mimicked the addition of mAb 3F10. Taken together, these data indicate that not all pools of AA are well used for LTC4 synthesis. These experiments also suggest that LTC4 synthesis in human basophils stimulated with fMLP depends on a SB 203347- and monoclonal antibody 3F10-inhibitable deacylation activity, presumably a sPLA2 acting at or near the cell surface. Furthermore, under normal conditions, this pool of AA is not observable because it is efficiently coupled to 5-lipoxygenase. Other deacylating enzymes, which do not supply AA for 5-lipoxygenase metabolism, also appear to be activated by fMLP and these other enzymes appear responsible for the net free AA normally observed after stimulation.
Footnotes
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Send reprint requests to: D.W. MacGlashan, Johns Hopkins Asthma & Allergy Ctr., 5501 Hopkins Bayview Circle, Baltimore, MD 21224.
- Abbreviations:
- AA
- arachidonic acid
- 5-HETE
- hydroxyeicosa-6,8,11,14-tetraenoic acid
- 5-HPETE
- hydroperoxyeicosa-6,8,11,14-tetraenoic acid
- GC/(NICI)MS
- gas chromatography negative ion chemical ionization mass spectrometry
- LTC4
- leukotriene C4
- PLA2
- phospholipase A2
- sPLA2
- secretory phospholipase A2
- 5-LO
- 5-lipoxygenase
- fMLP
- N-formyl-l-methionyl-l-leucyl-l-phenylalanine
- mAb 3F10
- monoclonal antibody 3F10
- cPLA2
- cytosolic phosholipase A2
- PG
- prostaglandin
- RBL
- rat basophilic leukemia cell
- rhPLA2
- recombinant human type II PLA2
- PIPES
- piperazine N,N′bis 2 ethane sulphonic acid
- BSA
- bovine serum albumin
- PMSF
- phenylmethlsulfonyl fluoride
- HSA
- human serum albumin
- FCS
- fetal calf serum
- ELISA
- enzyme linked immunosorbent assay
- BSTFA
- N,O-bis(trimethylsilyl)trifluoroacetamide
- PAG
- PIPES-albumin-glucose
- HPLC
- high-performance liquid chromatography
- Received September 10, 1996.
- Accepted November 28, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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