Protease activated receptor-4 in murine hepatic portal vein

Patrick Musami, Mandy Skasick, James D. Moffat*, Paul L. Andrews, and Iain Greenwood (Division of Basic Medical Sciences, St Georges, University of London, London, SW17 0RE; *Institute of Respiratory Diseases, Kings College London)

Protease-activated receptors (PAR1-4) are a heptahelical G-protein coupled receptor superfamily activated by thrombin, trypsin and other proteases that cleave the amino terminus on the extracellular domain. Whilst there have been a number of studies on PARS in vascular tissues there is no information on these receptors in the hepatic portal vein, a blood vessel that contracts spontaneously in situ and in vitro. The aim of the study was to investigate whether PAR agonists could influence the spontaneous contractile activity of whole hepatic portal veins.

Male Balb-C mice (8-11 wks of age; 20-40g) and male Sprague-Dawley rats (200-250g) were sacrificed according to Schedule 1 guidelines. Hepatic portal veins (hPV) were ligated in situ, removed and mounted in organ baths containing Krebs’ solution maintained at 37º C and gassed with 95% O 2/5% CO2.The resting tension applied was 0.1g(mice) and 0.5g(rats). The data was acquired using BIOPAC and Acquire Knowledge software (version 3.7). In all experiments, 60mM KCl was administered prior to the challenge with PAR agonists to test viability and establish the maximum contraction of the tissues. All data are the mean of n tissues ± s.e.m.

Application of trypsin to mouse hPV (10 U ml-1) produced a marked increase in contractile activity manifest as an increase in baseline of 0.09 ± 0.01g and a reduction in contractile interval by 50 ± 4 % (n=8). These effects were concentration-dependent across the range 0.5 to 10 U ml-1 and were also observed in rat hPV (n=4). Similar effects were also produced by 1 U ml-1 thrombin. The response to 10 U ml-1 trypsin was markedly attenuated by 5 min application of trypsin (0.5 to 10 U ml-1) 30 mins prior to the test challenge. The effect of thrombin was also decreased considerably after prior application of 10 U ml-1 trypsin. In contrast, repeated applications of α1- adrenoceptor agonist phenylepherine (10 μM) were not significantly different. Application of the PAR4 activating peptide AYP-GFK-NH2 (1 and 10 μM) markedly increased contractile activity (mean decrease in contractile interval produced by 10 μM was 32.5 % ± 6.07, n=4). However, AYP-GFK-NH2 did not affect the basal tension. The PAR1 and PAR2 activating peptides SFLLR-NH2 and SLIGRL-NH2 (10 and 100μM respectively) had no effect on any parameter of the spontaneous contractions.

These results suggest PAR 4 is present in the hepatic portal vein from mouse and rat and activation of this receptor has profound effects on the inherent rhythmicity of this blood vessel. Future studies will identify endogenous ligands for this receptor in this unique blood vessel.