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Inhibition by adenosine 3':5'-monophosphate of eicosanoid and platelet-activating factor biosynthesis in the mouse
B J Undem1, T J Torphy, D Goldman
1Division of Clinical Immunology, Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland 21224.
Abstract:
A mouse spleen-derived mast cell line (PT-18) was employed to examine the mechanisms of adenosine 3':5'-monophosphate (cAMP)-mediated inhibition of antigen-induced lipid mediator biosynthesis. Specifically, we tested the hypothesis that increasing cAMP in mast cells inhibits lipid mediator biosynthesis by a mechanism independent of effects on histamine release (degranulation) or changes in cytosolic calcium concentration. Forskolin inhibited antigen-induced prostaglandin D2 (PGD2), leukotriene C4 (LTC4), and leukotriene B4 (LTB4) production by 30-50%. In contrast, forskolin had no inhibitory effect on antigen-induced increases in cytosolic calcium concentration, as monitored by the calcium indicator fura-2, or histamine release from the cells. The combination of the phosphodiesterase inhibitor isobutylmethylxanthine with forskolin inhibited the antigen-induced production of PGD2 and LTC4 by 90-100% and histamine release by about 60%. These responses were accompanied by a virtual abolition of the antigen-induced increase in cytosolic calcium. To test further the hypothesis that increasing cAMP can lead to inhibition of lipid mediator biosynthesis in the absence of effects on cytosolic calcium, we employed the calcium ionophores A23187 and ionomycin. Forskolin alone or in combination with isobutylmethylxanthine had no effect on ionophore-induced increases in cytosolic calcium but effectively inhibited leukotriene biosynthesis. In addition, increasing cyclic AMP led to an inhibition of ionophore-induced production of platelet-activating factor and liberation of arachidonic acid. These data suggest that a relatively modest increase in cAMP-dependent protein kinase activity in mast cells leads to inhibition of the lipase-catalyzed cleavage of arachidonic acid from membrane phospholipids in the absence of measurable effects on either histamine release or changes in cytosolic calcium concentration. This effect results in a selective inhibition of the biosynthesis of lipid mediators including LTC4, LTB4, PGD2, and platelet-activating factor.
Insights
Increasing cyclic adenosine monophosphate (cAMP) in mast cells inhibits lipid mediator synthesis independently of histamine release or calcium changes. This points to a specific mechanism targeting arachidonic acid release.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Mast cells play a key role in allergic responses by releasing lipid mediators.
- Adenosine 3':5'-monophosphate (cAMP) is known to modulate mast cell function.
- The precise mechanisms by which cAMP influences lipid mediator biosynthesis are not fully understood.
Purpose of the Study:
- To investigate the role of cAMP in regulating antigen-induced lipid mediator biosynthesis in mast cells.
- To determine if cAMP-mediated inhibition occurs independently of histamine release and cytosolic calcium concentration changes.
- To elucidate the specific molecular targets of cAMP action in mast cells.
Main Methods:
- Utilized a mouse mast cell line (PT-18) for experiments.
- Administered forskolin to increase intracellular cAMP levels.
- Measured antigen-induced production of prostaglandin D2 (PGD2), leukotriene C4 (LTC4), and leukotriene B4 (LTB4).
- Monitored cytosolic calcium concentration using fura-2 and employed calcium ionophores (A23187, ionomycin).
- Assessed histamine release and platelet-activating factor production.
Main Results:
- Forskolin inhibited PGD2, LTC4, and LTB4 production by 30-50% without affecting histamine release or calcium levels.
- Combined treatment with forskolin and isobutylmethylxanthine (a phosphodiesterase inhibitor) nearly abolished PGD2 and LTC4 production and partially inhibited histamine release, with a significant reduction in calcium increase.
- Forskolin inhibited leukotriene biosynthesis, platelet-activating factor production, and arachidonic acid liberation induced by calcium ionophores, independent of direct effects on calcium concentration.
- These effects were associated with increased cAMP-dependent protein kinase activity.
Conclusions:
- Elevated cAMP levels in mast cells inhibit lipid mediator biosynthesis through a mechanism independent of histamine release or significant changes in cytosolic calcium.
- The primary inhibitory effect appears to target the lipase-catalyzed cleavage of arachidonic acid from phospholipids.
- This pathway selectively suppresses the synthesis of various lipid mediators, including LTC4, LTB4, PGD2, and platelet-activating factor.