Abstract
Because thrombin-induced inflammation is partially mast cell-dependent and involves proteinase-activated receptors (PARs), we hypothesized that mast cells express PAR and can be stimulated with PAR-activating peptides (PAR-AP). We demonstrated that rat peritoneal mast cells expressed PAR-1 and PAR-2 mRNA, and that PAR-2AP (tc-LIGRLO-NH2, 1 μM) induced 64.2 ± 4.4% specific β-hexosaminidase release from peritoneal mast cells, whereas another PAR-2AP (SLIGRL-NH2, 10 μM), trypsin (40 U/ml), and mast cell tryptase (1.5 μg/ml) did not. PAR-1AP (ApfFRChaCitY-NH2, 10 μM) (Cit) induced 11.7 ± 3.7% specific β-hexosaminidase release, whereas another PAR-1AP (TFLLR-NH2, 40 μM) and human thrombin (10 U/ml) did not. PAR-AP, tc-LIGRLO-NH2, and Cit increased the free intracellular Ca2+ concentration, whereas trypsin, tryptase, thrombin, and other PAR-APs did not. Desensitization of Ca2+ flux with different agonists suggests that although tc-LIGRLO-NH2, Cit, and compound 48/80 have similar mechanisms of action, tc-LIGRLO-NH2 also activates mast cells by a mechanism distinct from that of 48/80. Using benzalkonium chloride, which antagonizes the actions of 48/80 by competing for the same Gi protein, we determined that benzalkonium chloride suppressed tc-LIGRLO-NH2-mediated (0.1 μM) β-hexosaminidase release by 62%. Moreover, removal of sialic acid from peritoneal mast cells, using neuraminidase (2 U/ml), inhibited Cit- (10 μM, 52%) and tc-LIGRLO-NH2 (0.5 μM, 29%)-mediated β-hexosaminidase release. Thus, tc-LIGRLO-NH2 and Cit have at least partially similar mechanisms of action as 48/80. PAR-AP may therefore activate mast cells via multiple mechanisms that are distinct from those of classical PAR-1 and PAR-2. The responsiveness of mast cells to PAR-AP via a non-PAR-1/non-PAR-2 mechanism complicates the interpretation of in vivo studies using these peptides.
Footnotes
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This work was supported by funds from the Canadian Institutes for Health Research (to A.D.B. and M.D.H.) and a Postdoctoral Fellowship from the Canadian Lung Association/Medical Research Council of Canada/GlaxoWellcome (to G.R.S.). A.D.B. is the holder of the AstraZeneca Canada, Inc. (Chair in Asthma Research); J.L.W. is an Alberta Heritage Foundation for Medical Research Scientist and a Canadian Institutes for Health Research Senior Scientist; R.M. is an Alberta Heritage Foundation for Medical Research Senior Scholar; and H.V. is a Canadian Institutes for Health Research Scholar and an Alberta Heritage Foundation for Medical Research Clinical Investigator.
- Abbreviations:
- PAR
- proteinase-activated receptor
- PAR-AP
- proteinase-activated receptor-activating peptide
- RT
- reverse transcriptase
- PCR
- polymerase chain reaction
- HEK
- human embryonic kidney
- PBS
- phosphate-buffered saline
- HTB
- HEPES-Tyrode's buffer
- RT-PCR
- reverse transcription-polymerase chain reaction
- bp
- base pair(s)
- [Ca2+]i
- free intracellular calcium concentration
- Cit
- ApfFRChaCitY-NH2
- Received December 18, 2001.
- Accepted April 9, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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