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| Specific Observation(s) |
Source of Data |
| More than additive interaction of C5a and UDP in calcium release assays. |
RAW 2-Ligand Screen: Calcium
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| More than additive interaction of C5a and UDP in calcium release assays. |
Supplemental PDF
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| Abstract |
| C5a, a complement component derived from the C5 alpha chain, is a chemoattractant for neutrophils and macrophages. C5a acts through a G protein-coupled receptor to stimulate release of stored calcium in RAW264.7 cells. While the C5a receptor can stimulate members of both the Gi and Gq (G15/16) classes of heterotrimeric G proteins, the stimulation of calcium in RAW 264.7 cells is sensitive to pertussis toxin (PTX)(data not shown) and thus mediated primarily by Gi proteins. P2Y purinergic receptors are also G protein-coupled and mediate stimulation by UDP. The presumed pathway for stimulation of calcium in RAW 264.7 cells by UDP uses members of the Gq class of G proteins because this effect is insensitive to pertussis toxin.
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| The simultaneous application of C5a and UDP to RAW 264.7 cells causes an increase in calcium that is 50% greater than predicted by linear addition of increases in calcium obtained with the ligands applied alone. All of these responses are independent of extracellular calcium at the early times where potentiation occurs. This potentiation is not observed with platelet activating factor, which presumably stimulates calcium in a G protein-dependent, PTX- independent fashion. Two putative mechanisms to account for the synergism between UDP and C5a are suggested.
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| 1. Both pathways act on the same phospholipase C (PLC) isoform to produce greater than additive production of inositol 1,4,5-triphosphate (IP3) and subsequent release of calcium. PLC-beta isozymes have independent binding/activation sites for G-alpha-q (from UDP) and Gi beta-gamma subunits (from C5a) that when concomitantly occupied might produce synergistic effects on enzyme activity. PLC-beta 3 [Smrcka and Sternweis (1993) Biol.Chem. 268:9667-9674] is a potential candidate found in RAW 264.7 cells. |
| 2. Generation of IP3 and release of calcium are non-linear responses. Subthreshold stimulation by the slower response of C5a (peak response at 15-20 sec) at early times is amplified by the earlier stimulation of UDP (peak response at 10 sec or less). An argument against this mechanism is the lack of greater than additive effect of C5a with platelet activating factor, another robust stimulator of calcium in RAW 264.7 cells that is not affected by PTX. |
| References |
Smrcka AV and Sternweis PC (1993) Regulation of purified subtypes of phosphatidylinositol-specific phospholipase C beta by G protein alpha and beta gamma subunits. J Biol Chem. 268(13), 9667-9674.
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| Suggested Future Experiments |
| 1. Attempt to block the synergistic effect with PTX. If synergism is not blocked, the suggested input from Gi would be negated. Attenuation by PTX would not distinguish between the hypotheses, but would rule out the use of other G protein pathways by C5a. |
| 2. Measure IP3. With hypothesis 1, generation of this second messenger should be greater than additive. An additive response would be consistent with hypothesis 2 in the case of a non-linear potentiation between IP3 and calcium. |
| 3. Examine dose-response relationships. Amplification of signals between low doses of UDP that are "subthreshold" and C5a would support hypothesis 2. |
| 4. Knockdown specific PLC-beta isoforms. Elimination of stimulation by one ligand and not the other with knockdowns of specific phospholipases would suggest convergence of action after PLC (negate hypothesis1). In contrast, the attenuation of both stimuli by knockdown of a specific PLC isoform would be consistent with convergence before or at this molecule. |
| Discussion |
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