This study investigates, whether in addition to the protease‐activated receptor‐1 (PAR‐1), PAR‐4 is present in vascular smooth muscle cells (SMC) of the human saphenous vein and whether this receptor is functionally active. PAR‐1 and PAR‐4 are stimulated by thrombin and by the synthetic peptides SFLLRN and GYPGQV, respectively. mRNAs for both, PAR‐1 and PAR‐4, were detected in the SMC by using reverse transcriptase polymerase chain reaction (RT – PCR). Treatment of the SMC with GYPGQV (200 μM) resulted in a transient increase in free intracellular calcium. This calcium signal was completely abolished after a preceding challenge with thrombin (10 nM), indicating homologous receptor desensitization. Stimulation of the SMC with 10 nM thrombin or 200 μM SFLLRN caused a time‐dependent activation of the extracellular signal‐regulated kinases‐1/2 (ERK‐1/2) with a maximum at 5 min. In contrast, 100 nM thrombin as well as 200 μM of GYPGQV induced a prolonged phosphorylation of ERK‐1/2 with a maximum at 60 min. These data suggest that PAR‐1 and PAR‐4 are activated by thrombin at distinct concentrations and with distinct kinetics. GYPGQV stimulated [3H]‐thymidine incorporation in SMC. At 500 μM, the peptide increased DNA synthesis 2.5 fold above controls. A comparable mitogenic effect was obtained after stimulation of the SMC by 10 nM thrombin or 100 μM SFLLRN, respectively. These data indicate that a functionally active PAR‐4 is present in SMC and, in addition to PAR‐1, might contribute to thrombin‐induced mitogenesis. British Journal of Pharmacology (2001) 132, 1441–1446; doi:

1 The endothelial 5-HT receptor mediating relaxation of pig pulmonary artery has been characterized using the selective 5-HT 2B receptor agonist BW 723C86 and a variety of structurally diverse 5-HT receptor antagonists. 2 If arterial rings with intact endothelium were precontracted with prostaglandin F 2a (3 mM), BW 723C86 caused concentration-dependent relaxation with a pEC 50 =8.21+0.03 and E max =89+4% relative to 5-HT. The relaxant responses to BW 723C86 were inhibited by the 5-HT 2B receptor antagonist SB 204741, the 5-HT 2B/2C receptor antagonist SB 206553 and the antimigraine drug pizotifen, yielding pA 2 values of 6.68, 7.20 and 8.32, respectively. The pA 2 values against BW 723C86 were similar to those determined against 5-HT. 3 The relaxant eect of 5-HT was antagonized by a variety of 22 compounds of diverse chemical structures. Based on the calculated mean pA 2 values the order of the most potent antagonists was ritanserin (9.38) 4 methysergide (8.86) 4 pizotifen (8.47) 5 methiothepin (8.32) 4 LY 53857 (7.84) 5 amoxapine (7.80) 5 loxapine (7.73) 5 metergoline (7.64) 5 mianserin (7.51) 5 rauwolscine (7.39). Compounds with weak blocking potency were yohimbine (6.37), spiperone (5.88) and ketanserin (5.85). Correlation analysis between the anities of the antagonists in pig pulmonary artery and those from radioligand binding studies at human and rat 5-HT 2B receptors showed a highly signi®cant correlation (r=0.95 and 0.84, P50.002 and 50.005). Correlation with 5-HT 2C receptors was much lower (r=0.57, P=0.035), and no correlations were obtained with 5-ht 6 and 5-HT 7 receptors. 4 It is concluded that the 5-HT receptor mediating endothelium-dependent relaxation of pig pulmonary artery is of the 5-HT 2B subtype.

In PGF2 alpha-precontracted pulmonary arteries with intact endothelium, 5-hydroxytryptamine (5-HT, 1.0-100 nmol/l) caused a concentration-dependent reversible relaxation, at higher concentrations the contractile response prevailed. In endothelium-denuded vessels relaxation was absent. 5-HT-induced relaxation of precontracted pulmonary arteries was probably mediated by release of an endothelium-derived relaxing factor (EDRF). Preincubation of the arteries with methylene blue or NG-nitro-L-arginine (200 mumol/l) attenuated the relaxant effect. The 5-HT-induced relaxation was accompanied by an increase in cGMP. Indomethacin (3 mumol/l) did not influence the 5-HT-induced relaxation indicating that eicosanoids are not involved in the relaxant response to 5-HT. The 5-HT1C and 5-HT2 receptor agonist alpha-methyl-5-HT was as potent as 5-HT in inducing relaxation. The rank order of relaxant potency of the agonists investigated was alpha-methyl-5-HT > 5-HT > 5-methoxytryptamine > tryptamine > omega-methyl-5-HT > 5-carboxamidotryptamine > 2-methyl-5-HT > 5,6-dihydroxytryptamine > m-chlorophenylpiperazine > sumatriptan > 8-OH-DPAT. Phentolamine, pindolol and ICS 205-930 did not interfere with the relaxant effect. The 5-HT2 receptor antagonist ketanserin (1 mumol/l) inhibited the contractile response but did not alter vasodilatation. Apart from the blockade of the contractile effects, mesulergine, cyproheptadine and mianserin (0.1-3.0 mumol/l, each) induced a parallel shift to the right of the concentration-response curve for the relaxation induced by alpha-methyl-5-HT or 5-HT. Spiperone (0.3 mumol/l) exerted weak inhibitory effects on relaxation and contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

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1 This study investigates, whether in addition to the thrombin receptor (PAR-1), the proteinaseactivated receptor-2 (PAR-2) is present in vascular smooth muscle cells (SMC) and mediates mitogenesis. PAR-2 is activated by low concentrations of trypsin and the synthetic peptide SLIGRL. 2 Stimulation of bovine coronary artery SMC by trypsin (2 nM) caused a 3 fold increase in DNLA-synthesis. A similar e ect was observed with 10 nM thrombin. Trypsin-induced mitogenesis was inhibited by soybean trypsin inhibitor, indicating that the proteolytic activity of the enzyme was required for its mitogenic e ect. 3 The speci®c PAR-2-activating peptide SLIGRL or the PAR1-activating peptide SFFLRN did not elicit mitogenesis. 4 When the SMC were exposed to SLIGRL (40 nM), a homologous desensitization of cytosolic Ca 2+ mobilization was found after subsequent stimulation with trypsin (40 nM) but not thrombin (15 nM). 5 Trypsin (2 nM) as well as SLIGRL (100 mM) activated the nuclear factor kB (NFkB) with a maximum response 2 h after stimulation of the SMC. This suggests that both agonists acted via a common receptor, PAR-2. Maximum activation of NFkB by thrombin (10 nM) was detected after 4 ± 5 h. 6 These data suggest that PAR-2 is present in coronary SMC and mediates a mitogenic response. Activation of NFkB via either PAR-1 or PAR-2 does not predict mitogenesis.