Dual endothelium-dependent vascular activities of proteinase-activated receptor-2-activating peptides: evidence for receptor heterogeneity

Abstract

1. The vascular actions of the proteinase-activated receptor-2-activating peptides (PAR₂APs), SLIGRL-NH₂ (SL-NH₂) and SLIGKV-NH₂ (KV-NH₂) as well as the reverse-sequence peptide, LSIGRL-NH₂ (LS-NH₂) and an N-acylated PAR₂AP derivative, trans-cinnamoyl-LIGRLO-NH₂ (tcLI-NH₂), were studied in rat intact and endothelium-denuded artery ring preparations, primarily from the pulmonary artery (RPA).

2. In RPA rings with but not without a functional endothelium, SL-NH₂ (but not LS-NH₂) caused either an endothelium-dependent relaxation (at concentrations: <10 μM) or (at higher concentrations: >10 μM), an endothelium-dependent contraction. No contractile response was observed in endothelium-denuded preparations, that otherwise contracted in response to the PAR₁AP, TFLLR-NH₂.

3. The endothelium-dependent contractile response to SL-NH₂ was not blocked by the α-adrenoceptor antagonist prazosin, the endothelin antagonist BQ123, the angiotensin II antagonist DuP753, by tetrodotoxin; nor by the enzyme inhibitors, N^ω-nitro-L-arginine-methylester (NO-synthase), indomethacin (cyclo-oxygenase), SKF-525A (epoxygenase) and MK886 (leukotriene synthesis inhibitor).

4. In the relaxation assay, KV-NH₂ was 5 fold less potent than SL-NH₂, whereas in the contractile assay KV-NH₂ was about equipotent with SL-NH₂. However, the maximal contractile response to KV-NH₂ was lower than that of SL-NH₂.

5. The PAR₂AP analogue, tcLI-NH₂, was as active as SL-NH₂ in the relaxation assay but was inactive as a contractile agonist in the endothelium-intact RPA.

6. The relaxant responses caused by SL-NH₂ and trypsin, as well as the contractile response caused by SL-NH₂, did not desensitize in the course of repeated exposures of the tissue to agonist; whereas the contractile response to trypsin, only observed at concentrations greater than 30 u ml⁻¹, was desensitized by previous exposure of the tissue to either thrombin or trypsin.

7. In a contractile assay, where the tissue was desensitized to a concentration of trypsin that would otherwise cause a relaxant response, the preparation still contracted in response to SL-NH₂. However, the trypsin-desensitized preparations were no longer contracted by thrombin.

8. From the cross-desensitization by thrombin of the contractile response to trypsin (and vice versa), we concluded that the contractile effect of trypsin was due to activation of the thrombin receptor and not PAR₂.

9. We concluded that the endothelium-dependent contraction caused by high concentrations of SL-NH₂ is due to an as yet unidentified contracting factor; whereas the endothelium-dependent relaxation response observed at low concentrations of SL-NH₂ (⩽10 μM) is mediated by nitric oxide.

10. The distinct structure activity profiles for the contractile response (potency of KV-NH₂⩽SL-NH₂) compared with the relaxant response (potency of KV-NH₂<5SL-NH₂); the contractile responsiveness to SL-NH₂ of an endothelium-intact RPA preparation, that did not contract in response to trypsin; and the lack of contractile activity of the PAR₂AP analogue tcLI-NH₂, that was as active as SL-NH₂ in the relaxation assay all argue in favour of receptor heterogeneity in the vasculature for the PAR₂APs. It remains to be determined if the distinct endothelial receptor responsible for the contractile action of SL-NH₂ can be proteolytically activated, like PAR₁ and PAR₂.

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