Regulation of arachidonic acid, eicosanoid, and phospholipase A2 levels in murine mast cells by recombinant stem cell factor.
The current study evaluates the capacity of recombinant rat stem cell factor (rrSCF) to regulate enzymes that control AA release and eicosanoid generation in mouse bone marrow-derived mast cells (BMMCs). Initial studies indicated that rrSCF provided for 24 h inhibited the release of AA into supernatant fluids of antigen- and ionophore A23187-stimulated BMMCs. Agonist-induced increases in cellular levels of AA were also inhibited, albeit to a lesser degree by rrSCF. To determine the inhibitory mechanism, several steps (e.g., mobilization of cytosolic calcium, release of BMMC granules, and regulation of phospholipase A2 [PLA2] activity) that could influence AA release were measured in rrSCF-treated cells. rrSCF did not alter the capacity of BMMCs to mobilize cytosolic calcium or release histamine in response to antigen and ionophore. BMMCs released large amounts of PLA2 with characteristics of the group II family in response to antigen and ionophore A23187. rrSCF treatment of BMMCs reduced the secretion of this PLA2 activity by BMMCs. Partial purification of acid-extractable PLA2 from rrSCF-treated and untreated BMMCs suggested that rrSCF decreased the quantity of acid-stable PLA2 within the cells. In contrast to group II PLA2, the quantity of cPLA2 (as determined by Western blot analysis) increased in response to rrSCF. To assess the ramifications of rrSCF-induced reductions in AA and group II PLA2, eicosanoid formation was measured in antigen- and ionophore-stimulated BMMCs, rrSCF-inhibited (100 ng/ml, 24 h) prostaglandin D2 (PGD2), thromboxane B2, and leukotriene B4 by 48.4 +/- 7.7%, 61.1 +/- 10.0% AND 38.1 +/- 3.6%, respectively, in antigen-stimulated cells. Similar patterns of inhibition were observed in ionophore-stimulated BMMCs. The addition of a group I PLA2 or exogenous AA to BMMCs reversed the inhibition of eicosanoid generation induced by rrSCF. Together, these data indicate that rrSCF differentially regulates group II and cytosolic PLA2 activities in BMMCs. The resultant reductions in eicosanoid generation suggest that group II PLA2 provides a portion of AA that is used for eicosanoid biosynthesis by BMMCs.
Fonteh
A N
AN
Department of Internal Medicine, Bowman Gray School of Medicine, Winston-Salem, North Carolina 27517, USA.
Samet
J M
JM
Chilton
F H
FH
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015777:Eicosanoids / Q000378:metabolism*; D005230:Fatty Acids, Nonesterified / Q000378:metabolism; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology*; D007700:Kinetics; D008407:Mast Cells / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008808:Mice, Inbred CBA; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D051381:Rats; D011994:Recombinant Proteins / Q000494:pharmacology; D019089:Stem Cell Factor; D013997:Time Factors
D015777:Eicosanoids; D005230:Fatty Acids, Nonesterified; D016298:Hematopoietic Cell Growth Factors; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D016718:Arachidonic Acid; D000001:Calcimycin; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium