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Derivation of ligands for the complement C3a receptor from the C-terminus of C5a.

Halai R, Bellows-Peterson ML, Branchett W, Smadbeck J, Kieslich CA, Croker DE, Cooper MA, Morikis D, Woodruff TM, Floudas CA, Monk PN - Eur. J. Pharmacol. (2014)

Bottom Line: No agonist/antagonist activity was observed at C5a1, but we instead saw that the ligands were able to partially agonize the closely related complement receptor C3a receptor.This was verified in the presence of C3a receptor antagonist SB 290157 and in a stable cell line expressing either C5a1 or C3a receptor alone.C3a agonism has been suggested to be a potential treatment of acute neutrophil-driven traumatic pathologies, and may have great potential as a therapeutic avenue in this arena.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.

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Agonist activity of peptides at human C5a1 and C3a receptors expressed in rat basophilic leukemia cells (RBL-2H3). Peptides were dissolved in DMSO and incubated at 50 μM with RBL-2H3 cells transfected with the appropriate receptor for 15 min. Degranulation was measured as the secretion of β-hexosaminidase. Results are expressed relative to maximal stimulation with 200 nM C5a (C5a1) or 100 nM hexapeptide agonist FLPLAR (C3a) after subtraction of background. Statistical significance of the difference from zero was assessed using a one-sample t test (⁎P<0.05; ⁎⁎P<0.001). Bars are shaded according to peptide length (see Supplementary Table 2).
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f0015: Agonist activity of peptides at human C5a1 and C3a receptors expressed in rat basophilic leukemia cells (RBL-2H3). Peptides were dissolved in DMSO and incubated at 50 μM with RBL-2H3 cells transfected with the appropriate receptor for 15 min. Degranulation was measured as the secretion of β-hexosaminidase. Results are expressed relative to maximal stimulation with 200 nM C5a (C5a1) or 100 nM hexapeptide agonist FLPLAR (C3a) after subtraction of background. Statistical significance of the difference from zero was assessed using a one-sample t test (⁎P<0.05; ⁎⁎P<0.001). Bars are shaded according to peptide length (see Supplementary Table 2).

Mentions: RBL-2H3 cells transfected with either human C5a1 or human C3a receptor (Cain and Monk, 2002) were used to confirm hits detected in HMDM, using degranulation as a read-out (Monk et al., 1994). In cells transfected with C5a1, very weak agonist activity was detected in two groups of peptides, although even at 100 µM (the maximal dose achievable in the presence of the DMSO, which has adverse effects >1%), the activation obtained was 5–10% of the maximum activation achieved by a high dose of C5a (Fig. 3, top panel). In contrast, two peptides (31 and 54) produced very strong activation of C3a receptor (Fig. 3, lower panel) at 100 µM. In antagonist assays using two different doses of C5a that caused 50% or 100% degranulation, none of the peptides had any antagonist activity, even when pre-incubated with cells at the maximum achievable dose, 100 µM (data not shown).


Derivation of ligands for the complement C3a receptor from the C-terminus of C5a.

Halai R, Bellows-Peterson ML, Branchett W, Smadbeck J, Kieslich CA, Croker DE, Cooper MA, Morikis D, Woodruff TM, Floudas CA, Monk PN - Eur. J. Pharmacol. (2014)

Agonist activity of peptides at human C5a1 and C3a receptors expressed in rat basophilic leukemia cells (RBL-2H3). Peptides were dissolved in DMSO and incubated at 50 μM with RBL-2H3 cells transfected with the appropriate receptor for 15 min. Degranulation was measured as the secretion of β-hexosaminidase. Results are expressed relative to maximal stimulation with 200 nM C5a (C5a1) or 100 nM hexapeptide agonist FLPLAR (C3a) after subtraction of background. Statistical significance of the difference from zero was assessed using a one-sample t test (⁎P<0.05; ⁎⁎P<0.001). Bars are shaded according to peptide length (see Supplementary Table 2).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263610&req=5

f0015: Agonist activity of peptides at human C5a1 and C3a receptors expressed in rat basophilic leukemia cells (RBL-2H3). Peptides were dissolved in DMSO and incubated at 50 μM with RBL-2H3 cells transfected with the appropriate receptor for 15 min. Degranulation was measured as the secretion of β-hexosaminidase. Results are expressed relative to maximal stimulation with 200 nM C5a (C5a1) or 100 nM hexapeptide agonist FLPLAR (C3a) after subtraction of background. Statistical significance of the difference from zero was assessed using a one-sample t test (⁎P<0.05; ⁎⁎P<0.001). Bars are shaded according to peptide length (see Supplementary Table 2).
Mentions: RBL-2H3 cells transfected with either human C5a1 or human C3a receptor (Cain and Monk, 2002) were used to confirm hits detected in HMDM, using degranulation as a read-out (Monk et al., 1994). In cells transfected with C5a1, very weak agonist activity was detected in two groups of peptides, although even at 100 µM (the maximal dose achievable in the presence of the DMSO, which has adverse effects >1%), the activation obtained was 5–10% of the maximum activation achieved by a high dose of C5a (Fig. 3, top panel). In contrast, two peptides (31 and 54) produced very strong activation of C3a receptor (Fig. 3, lower panel) at 100 µM. In antagonist assays using two different doses of C5a that caused 50% or 100% degranulation, none of the peptides had any antagonist activity, even when pre-incubated with cells at the maximum achievable dose, 100 µM (data not shown).

Bottom Line: No agonist/antagonist activity was observed at C5a1, but we instead saw that the ligands were able to partially agonize the closely related complement receptor C3a receptor.This was verified in the presence of C3a receptor antagonist SB 290157 and in a stable cell line expressing either C5a1 or C3a receptor alone.C3a agonism has been suggested to be a potential treatment of acute neutrophil-driven traumatic pathologies, and may have great potential as a therapeutic avenue in this arena.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.

Show MeSH
Related in: MedlinePlus