Endothelium-dependent contractile actions of proteinase-activated receptor-2-activating peptides in human umbilical vein: release of a contracting factor via a novel receptor

1. The contractile actions of the proteinase-activated receptor-2-activating peptides (PAR₂APs), SLIGRL-NH₂ (SL-NH₂), SLIGKV-NH₂ (KV-NH₂), trans-cinnamoyl-LIGRLO-NH₂ (tc-NH₂), and the PAR₁-AP, TFLLR-NH₂ (TF-NH₂) as well as trypsin and thrombin were studied in endothelium-denuded and intact human umbilical vein (HUV) ring preparations.
2. In HUV rings with, but not without an intact endothelium, PAR₂APs caused a concentration-dependent contractile response, whereas LSIGRL-NH₂ trypsin and PAR₁APs were inactive. The contractile response was not affected by the endothelin ET_A receptor antagonist, BQ123, the cyclooxygenase inhibitor, indomethacin, the leukotriene synthesis inhibitor, MK886, or the epoxygenase/P450 inhibitor, SKF-525A. Other pharmacological antagonists (prazosin, Losartan®) were similarly inactive.
3. The order of potencies of the PAR₂APs to cause a contraction in the endothelium-intact preparation was: SL-NH₂>>KV-NH₂⩾tc-NH₂.
4. Using an endothelium-free rat aorta ring as a reporter tissue, surrounded with endothelium-intact HUV as a donor tissue in a `sandwich assay,'' we also monitored the ability of SL-NH₂, TF-NH₂, trypsin and thrombin to release either contractile (EDCF) or relaxant (EDRF) factors.
5. In the `sandwich assay'' done in the presence of L-NAME (0.1 mM), the endothelium-intact HUV tissue (but not endothelium-denuded HUV) released a contractile factor (EDCF) in response to SL-NH₂ (50 μM) but not to trypsin or LSIGRL-NH₂. The SL-NH₂-mediated release/action of the EDCF was not affected by BQ123, indomethacin, MK886 or SKF-525A.
6. In the `sandwich assay'', trypsin (4–10 nM), SL-NH₂, KV-NH₂ and tc-NH₂ caused the release of a relaxant activity (EDRF) from the endothelium-intact (but not the denuded) HUV preparation. The release of EDRF was blocked by 0.1 mM ^ωnitro-L-arginine-methylester (L-NAME). Neither thrombin (10 u ml⁻¹, 100 nM) nor TF-NH₂ (50 μM) were active in this EDRF-release assay.
7. The relative potencies of the PAR₂ agonists for causing the release of EDRF in the HUV sandwich assay were: trypsin>>SL-NH₂>>tc-NH₂>KV-NH₂. This order of potencies differed from the one observed for the same agonists in the HUV contraction assay (above) and in an intracellular calcium signalling assay, conducted with cloned human PAR₂ that was expressed in cultured rat kidney KNRK cells: trypsin>>SL-NH₂=tc-NH₂>KV-NH₂.
8. We conclude that PAR₂APs (but not PAR₁APs) via a receptor distinct from PAR₂, can cause a contractile response in endothelium-intact HUV tissue via the release of a diffusable EDCF, that is different from previously recognized smooth muscle agonists (e.g. prostanoid metabolites, endothelin, noradrenaline, angiotensin-II, acetylcholine).