A peptide analogue of thrombin receptor-activating peptide inhibits thrombin and thrombin-receptor-activating peptide-induced vascular smooth muscle cell proliferation

The serine protease thrombin, in addition to its pivotal role in the coagulation cascade, plays an important role in the development of atherosclerosis and restenosis by inducing smooth cell proliferation. Thrombin exerts its cellular effects mainly by cleaving its own receptor, leaving a new NH2-terminus that can act as a tethered ligand to activate the thrombin receptor. Peptides derived from the new NH2-terminus are able to fully activate thrombin receptor and mimic cellular effects of thrombin. Peptides with structural similarities to the tethered ligand have been tested for their ability to prevent thrombin- and tethered ligand-induced platelet aggregation and thrombus formation. We synthesized a peptide with multiple alanine substitutions in both critical and noncritical residues of tethered ligand that specifically inhibited platelet aggregation induced by thrombin and thrombin receptor-activating peptide and prevented thrombus formation in a rabbit thrombosis model. In the present study we demonstrate that this peptide inhibited only thrombin- and tethered ligand-induced human vascular smooth muscle cell proliferation as determined by (3H)-thymidine incorporation and has no effect on platelet-derived growth factor and serum-induced smooth muscle cell proliferation. The inhibitory effect of this peptide is dependent on the concentration of the antagonist used and length of preincubation time. The possible mechanism by which this peptide exerts its inhibitory effect may by desensitizing the thrombin receptor. The results of the present study suggest that apart from being antithrombotic, tethered ligand antagonist peptides can also act as antiatherosclerotic or antirestenotic agents.     

Inhibition of cellular action of thrombin by N3-cyclopropyl-7-[[4-(1-methylethyl)phenyl]methyl]-7H-pyrrolo[3, 2-f]quinazoline-1,3-diamine (SCH 79797), a nonpeptide thrombin receptor antagonist

A growing body of evidence suggests an important contribution of the cellular actions of thrombin to thrombosis and restenosis following angioplasty. Recently we reported on SCH 79797 (N3-cyclopropyl-7-?[4-(1-methylethyl)phenyl]methyl?-7H-pyrrolo[3, 2-f]quinazoline-1,3-diamine) and its analogs as new potent, nonpeptide thrombin receptor antagonists. This study further characterizes the biochemical and pharmacological actions of pyrroloquinazoline inhibitors of protease activated receptor-1 (PAR-1) in human platelets and coronary artery smooth muscle cells (hCASMC). SCH 79797 and its N-methyl analog (SCH 203099) inhibited binding of a high-affinity thrombin receptor-activating peptide ([(3)H]haTRAP, Ala-Phe(p-F)-Arg-ChA-HArg-[(3)H]Tyr-NH(2)) to PAR-1 with IC(50) values of 70 and 45 nM, respectively. SCH 79797 inhibited [(3)H]haTRAP binding in a competitive manner. SCH 79797 and SCH 203099 inhibited alpha-thrombin- and haTRAP-induced aggregation of human platelets, but did not inhibit human platelet aggregation induced by the tethered ligand agonist for protease-activated receptor-4 (PAR-4), gamma-thrombin, ADP, or collagen. SCH 203099 inhibited surface expression of P-selectin induced by haTRAP and thrombin, and it did not increase P-selectin expression or prevent thrombin cleavage of the receptor. Thrombin and TFLLRNPNDK-NH(2) (TK), a PAR-1-selective agonist, produced transient increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in hCASMC. This increase in [Ca(2+)](i) was inhibited effectively by SCH 79797. However, the Ca(2+) transients induced by SLIGKV-NH(2,) a PAR-2-selective agonist, were not inhibited by SCH 79797. Thrombin- and TK-stimulated [(3)H]thymidine incorporation also was inhibited completely by SCH 79797. The results of this study demonstrate that SCH 79797 and SCH 203099 are potent, selective antagonists of PAR-1 in human platelets and hCASMC. These data also suggest that the thrombin stimulation of Ca(2+) transients and mitogenesis in hCASMC is mediated primarily through activation of PAR-1.     

Bronchoconstrictor effect of thrombin and thrombin receptor activating peptide in guinea-pigs in vivo

1. Several thrombin cellular effects are dependent upon stimulation of proteinase activated receptor-1 (PAR-1) localized over the cellular surface. Following activation by thrombin, a new N-terminus peptide is unmasked on PAR-1 receptor, which functions as a tethered ligand for the receptor itself. Synthetic peptides called thrombin receptor activating peptides (TRAPs), corresponding to the N-terminus residue unmasked, reproduce several thrombin cellular effects, but are devoid of catalytic activity. We have evaluated the bronchial response to intravenous administration of human alpha-thrombin or a thrombin receptor activating peptide (TRAP-9) in anaesthetized, artificially ventilated guinea-pigs. 2. Intravenous injection of thrombin (100 microkg(-1)) caused bronchoconstriction that was recapitulated by injection of TRAP-9 (1 mg kg(-1)). Animal pretreatment with the thrombin inhibitor Hirulog (10 mg kg(-1) i.v.) prevented thrombin-induced bronchoconstriction, but did not affect bronchoconstriction induced by TRAP-9. Both agents did not induce bronchoconstriction when injected intravenously to rats. 3. The bronchoconstrictor effect of thrombin and TRAP-9 was subjected to tolerance; however, in animals desensitized to thrombin effect, TRAP-9 was still capable of inducing bronchoconstriction, but not vice versa. 4. Depleting animals of circulating platelets prevented bronchoconstriction induced by both thrombin and TRAP-9. 5. Bronchoconstriction was paralleled by a biphasic change in arterial blood pressure, characterized by a hypotensive phase followed by a hypertensive phase. Thrombin-induced hypotension was not subject to tolerance and was inhibited by Hirulog; conversely, hypertension was subject to tolerance and was not inhibited by Hirulog. Hypotension and hypertension induced by TRAP-9 were neither subject to tolerance nor inhibited by Hirulog. 6. Our results indicate that thrombin causes bronchoconstriction in guinea-pigs through a mechanism that requires proteolytic activation of its receptor and the exposure of the tethered ligand peptide. Platelet activation might be triggered by the thrombin effect.     

Thrombin receptor antagonists; recent advances in PAR-1 antagonist development

The receptor for the serine protease thrombin, the protease-activated receptor-1 (PAR-1), has been recently characterized. Its key roles in thrombin-stimulated human platelet activation, vascular endothelial and smooth muscle proliferation, inflammatory responses and neurodegeneration suggest receptor involvement in various disorders such as arterial thrombosis, atherosclerosis, restenosis, inflammation and myocardial infarction. It has been established that thrombin elicits the majority of its effects via PAR-1. PAR-1 has a novel mechanism of activation. The receptor, a member of the seven-transmembrane domain receptor family, is cleaved by thrombin at a specific site on the N-terminal extension, and a newly exposed N-terminus acts as a tethered ligand to activate the receptor itself. The need for development of a PAR-1 antagonist that may be valuable as a therapeutic agent has been recognized. An intriguing challenge is the necessity of the antagonist to compete with an intramolecular ligand while showing no intrinsic activity. The lead compounds were found to be synthetic peptides containing N-terminal hexapeptide or pentapeptide (Ser-Phe-Leu-Leu-Arg-Asn, Ser-Phe-Leu-Leu-Arg) or modified sequences (TRAPs; thrombin receptor-activating peptides), which exhibit full PAR-1 agonist activity. Selective PAR-1 antagonists have already been synthesized. Though their potency is still not enough to justify therapeutic use, it is clear that future progress will bring a novel class of drugs-thrombin receptor antagonists. The emphasis of this review, therefore, will be placed on advances in the discovery of potent and selective PAR-1 antagonists.     

Proteinase-activated receptor-1 agonists attenuate nociception in response to noxious stimuli

Proteinase-activated receptor-1 (PAR-1) is activated by thrombin and can be selectively activated by synthetic peptides (PAR-1-activating peptide: PAR-1-AP) corresponding to the receptor's tethered ligand. PAR-1 being expressed by afferent neurons, we investigated the effects of PAR-1 agonists on nociceptive responses to mechanical and thermal noxious stimuli. Intraplantar injection of selective PAR-1-AP increased nociceptive threshold and withdrawal latency, leading to mechanical and thermal analgesia, while control peptide had no effect. Intraplantar injection of thrombin also showed analgesic properties in response to mechanical, but not to thermal stimulus. Co-injection of PAR-1-AP with carrageenan significantly reduced carrageenan-induced mechanical and thermal hyperalgesia, while thrombin reduced carrageenan-induced mechanical but not thermal hyperalgesia. The fact that thrombin is not a selective agonist for PAR-1 may explain the different effects of thrombin and PAR-1-AP. These results identified analgesic properties for selective PAR-1 agonists that can modulate nociceptive response to noxious stimuli in normal and inflammatory conditions.     

The novel and orally active thrombin receptor antagonist E5555 (Atopaxar) inhibits arterial thrombosis without affecting bleeding time in guinea pigs

Thrombin is a powerful agonist for platelets, the action of which is mediated by the thrombin receptor protease-activated receptor-1 (PAR-1). Recently, we discovered that E5555 (1-(3-tert-butyl-4-methoxy-5-morpholinophenyl)-2-(5,6-diethoxy-7-fluoro-1-imino-1,3-dihydro-2H-isoindol-2-yl) ethanone hydrobromide) is a potent thrombin receptor antagonist. We evaluated the anti-platelet and anti-thrombotic effects of E5555. E5555 inhibited the binding of a high-affinity thrombin receptor-activating peptide ([(3)H]haTRAP) to PAR-1 with a half maximal inhibitory concentration (IC(50)) value of 0.019μM. E5555 showed potent inhibitory effects on human platelet aggregation induced by thrombin and TRAP with IC(50) values of 0.064 and 0.031μM, respectively, but had no effect on platelet aggregation induced by either ADP or collagen. Similarly, E5555 showed potent and selective inhibitory effects on guinea pig platelet aggregation induced by thrombin and TRAP with IC(50) values of 0.13 and 0.097μM, respectively. The antithrombotic activity of E5555 in vivo was evaluated in a photochemically-induced thrombosis (PIT) model using guinea pigs. Oral administration of E5555 at 30 and 100mg/kg prolonged the time to occlusion by 1.8-fold and 2.4-fold, respectively, compared with controls. Furthermore, E5555 did not prolong bleeding time in guinea pigs at the highest tested dosage of 1000mg/kg. The drug interactions between E5555 and tissue plasminogen activator (tPA) were evaluated. Intravenous administration of 1mg/kg tPA significantly prolonged bleeding time, and its effects were not altered by the oral co-administration of 300mg/kg E5555. These results suggest that E5555 could be a therapeutic option for atherothrombotic disease.     

Proteinase-activated receptors

Proteinase-activated receptors are a recently described, novel family of seven-transmembrane G-protein-coupled receptors. Rather then being stimulated through ligand receptor occupancy, activation is initiated by cleavage of the N terminus of the receptor by a serine protease resulting in the generation of a new tethered ligand that interacts with the receptor within extracellular loop-2. To date, four proteinase-activated receptors (PARs) have been identified, with distinct N-terminal cleavage sites and tethered ligand pharmacology. In addition to the progress in the generation of PAR-1 antagonists, we describe the role of thrombin in such processes as wound healing and the evidence implicating PAR-1 in vascular disorders and cancer. We also identify advances in the understanding of PAR-1-mediated intracellular signaling and receptor desensitization. The cellular functions, signaling events, and desensitization processes involved in PAR-2 activation are also assessed. However, other major aspects of PAR-2 are highlighted, in particular the ability of several serine protease enzymes, in addition to trypsin, to function as activators of PAR-2. The likely physiological and pathophysiological roles for PAR-2 in skin, intestine, blood vessels, and the peripheral nervous system are considered in the context of PAR-2 activation by multiple serine proteases. The recent discovery of PAR-3 and PAR-4 as additional thrombin-sensitive PARs further highlights the complexity in assessing the effects of thrombin in several different systems, an issue that remains to be fully addressed. These discoveries have also highlighted possible PAR-PAR interactions at both functional and molecular levels. The future identification of other PARs and their modes of activation are an important future direction for this expanding field of study.     

Oral administration of the thrombin receptor antagonist E5555 (atopaxar) attenuates intimal thickening following balloon injury in rats

Thrombin is a powerful agonist for a variety of cellular responses including platelet aggregation and vascular smooth muscle cell (SMC) proliferation. These actions are mediated by a thrombin receptor known as protease-activated receptor-1 (PAR-1). Recently we discovered that 1-(3-tert-butyl-4-methoxy-5-morpholinophenyl)-2-(5,6-diethoxy-7-fluoro-1-imino-1,3-dihydro-2H-isoindol-2-yl)ethanone hydrobromide (E5555, atopaxar) is a potent and selective thrombin receptor antagonist. This study characterized the pharmacological effects of E5555 on SMC proliferation in vitro and in a rat model of intimal thickening after balloon injury in vivo. E5555 selectively inhibited rat aortic SMC proliferation induced by thrombin and thrombin receptor-activating peptide (TRAP) with half maximal inhibitory concentration (IC(50)) values of 0.16 and 0.038 μM, respectively. E5555 did not inhibit rat SMC proliferation induced by basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) at concentrations up to 1μM. In addition, E5555 inhibited human aortic SMC proliferation induced by thrombin at concentrations of 0.3 and 3units/ml with IC(50) values of 0.028 and 0.079 μM, respectively, whereas it did not affect bFGF-induced proliferation at concentrations up to 1μM. Repeated oral administration of 30 mg/kg E5555 (once daily for 16 days) significantly reduced neointimal formation in the balloon-injured rat arterial model. These results suggested that a PAR-1 antagonist could be effective for treating restenosis following vascular intervention in addition to preventing thrombus formation. E5555 could thus have therapeutic potential for restenosis and chronic atherothrombotic disease.     

凝血酶受体拮抗剂在抗血栓方面的研究进展

20世纪90年代初克隆和发现的凝血酶受体(PAR-1)为研制抗血栓药物提供了一个新靶点,引起学术界和制药业的广泛关注。由于凝血酶受体的特殊活化机制,其自身相连的活化氮端与活化中心近在咫尺,只有结合力很高的小分子化合物才能有效地拮抗凝血酶受体。因此,多年来,只有少数化合物被发现具有较好的凝血酶受体拮抗活性,其中vorapaxar和atopaxar进入了临床试验。vorapaxar在Ⅲ期临床试验发现有显著疗效,但也有出血不良反应,尤其不适用于有中风史的患者。最近,vorapaxar与PAR-1结合的晶体结构已经发表。这些结果为设计和研制新一代凝血酶受体拮抗剂指出了优化的方向,提供了分子水平的结构信息。本文从药物化学角度综述近年来凝血酶受体拮抗剂的研究进展和现状,重点描述vorapaxar、atopaxar以及相关化合物和最新发表的PAR-1拮抗剂。     

Physiology of protease-activated receptors (PARs): involvement of PARs in digestive functions

The protease-activated receptor (PAR), a G protein-coupled receptor present on cell surface, mediates cellular actions of extracellular proteases. Proteases cleave the extracellular N-terminal of PAR molecules at a specific site, unmasking and exposing a novel N-terminal, a tethered ligand, that binds to the body of receptor molecules resulting in receptor activation. Amongst four distinct PARs that have been cloned, PARs 1, 3 and 4 are activated by thrombin, but PAR-2 is activated by trypsin or mast cell tryptase. Human platelets express two distinct thrombin receptors, PAR-1 and PAR-4, while murine platelets express PAR-3 and PAR-4. Apart from roles of PARs in platelet activation, PARs are distributed to a number of organs in various species, predicting their physiological importance. We have been evaluating agonists specific for each PAR, using multiple procedures including a HEK cell calcium signal receptor desensitization assay. Using specific agonists that we developed, we found the following: 1) the salivary glands express PAR-2 mRNA and secret saliva in response to PAR-2 activation; 2) pancreatic juice secretion occurs following in vivo PAR-2 activation; 3) PAR-1 and PAR-2 modulate duodenal motility. Collectively, PAR plays various physiological and/or pathophysiological roles, especially in the digestive systems, and could be a novel target for drug development.     

Effects of baicalein isolated from Scutellaria baicalensis Radix on adhesion molecule expression induced by thrombin and thrombin receptor agonist peptide in cultured human umbilical vein endothelial cells.

We examined the effects of various flavonoids isolated from the roots of Scutellaria baicalensis Georgi on adhesion molecule expression induced by thrombin and thrombin receptor agonist peptide (SFLLRNPNDKYEPF, TRAP) in cultured human umbilical vein endothelial cells. Thrombin and thrombin receptor agonist peptide induced endothelial leukocyte adhesion molecule-1 (ELAM-1) expression. Intercellular adhesion molecule-1 (ICAM-1) expression was also induced by thrombin, but not by TRAP. Baicalein isolated from Scutellariae Radix inhibited ELAM-1 expression induced by thrombin and thrombin receptor agonist peptide dose-dependently, with 50% inhibitory concentrations (IC50) of 5.53 +/- 1.68 microM and 2.44 +/- 1.08 microM, respectively. Furthermore, baicalein inhibited thrombin-induced ICAM-1 expression with an IC50 of 9.67 +/- 1.28 microM. In addition, baicalein inhibited the expressions of ELAM-1 and ICAM-1 stimulated by protein kinase C (PKC) activator phorbol myristate acetate (PMA).     

Structural features of the glutamate binding site in recombinant NR1/NR2A N-methyl-D-aspartate receptors determined by site-directed mutagenesis and molecular modeling

We have used site-directed mutagenesis of amino acids located within the S1 and S2 ligand binding domains of the NR2A N-methyl-D-aspartate (NMDA) receptor subunit to explore the nature of ligand binding. Wild-type or mutated NR1/NR2A NMDA receptors were expressed in Xenopus laevis oocytes and studied using two electrode voltage clamp. We investigated the effects of mutations in the S1 and S2 regions on the potencies of the agonists L-glutamate, L-aspartate, (R,S)-tetrazol-5yl-glycine, and NMDA. Mutation of each of the corresponding residues found in the NR2A receptor subunit, suggested to be contact residues in the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit, caused a rightward shift in the concentration-response curve for each agonist examined. None of the mutations examined altered the efficacy of glutamate as assessed by methanethiosulfonate ethylammonium potentiation of agonist-evoked currents. In addition, none of the mutations altered the potency of glycine. Homology modeling and molecular dynamics were used to evaluate molecular details of ligand binding of both wild-type and mutant receptors, as well as to explore potential explanations for agonist selectivity between glutamate receptor subtypes. The modeling studies support our interpretation of the mutagenesis data and indicate a similar binding strategy for L-glutamate and NMDA when they occupy the binding site in NMDA receptors, as has been proposed for glutamate binding to the GluR2 AMPA receptor subunit. Furthermore, we offer an explanation as to why "charge conserving" mutations of two residues in the binding pocket result in nonfunctional receptor channels and suggest a contributing molecular determinant for why NMDA is not an agonist at AMPA receptors.     

Multiple roles for protease-activated receptor-2 in gastric mucosa

Protease-activated receptor-2 (PAR-2), a G-protein-coupled receptor, is activated by proteolytic unmasking of the N-terminal cryptic tethered ligand. In gastric mucosa, activation of PAR-2 expressed by sensory neurons triggers release of CGRP and tachykinins, leading to mucus secretion via activation of CGRP₁ and NK₂ receptors, respectively. The PAR-2 agonist reveals mucosal cytoprotective effects in several gastric injury models. Further, PAR-2 activation inhibits gastric acid secretion, independently of sensory neurons or prostanoid formation. The PAR-2 agonist also induces increase in gastric mucosal blood flow, an effect being independent of endogenous CGRP or NO. Endothelium-derived hyperpolarizing factor (EDHF) appears to be involved in the PAR-2-mediated enhancement of mucosal blood flow. In contrast, mucosal chief cells are abundant in immunoreactive PAR-2, and PAR-2 stimulation triggers pepsinogen secretion. Taken together, primarily, PAR-2 plays protective roles in gastric mucosa through multiple mechanisms. Considering PAR-2-mediated pepsinogen secretion, PAR-2 might function as a double-edged sword in gastric mucosa.     

Development of agonists/antagonists for protease-activated receptors (PARs) and the possible therapeutic application to gastrointestinal diseases

Protease-activated receptors (PARs), a family of G-protein-coupled seven-transmembrane-domain receptors, are activated by proteolytic unmasking of the N-terminal cryptic tethered ligand by certain serine proteases. Among four PAR family members cloned to date, PAR-1, PAR-2, and PAR-4 can also be activated through a non-enzymatic mechanism, which is achieved by direct binding of exogenously applied synthetic peptides based on the tethered ligand sequence, known as PARs-activating peptides, to the body of the receptor. Various peptide mimetics have been synthesized as agonists for PARs with improved potency, selectivity, and stability. Some peptide mimetics and/or nonpeptide compounds have also been developed as antagonists for PAR-1 and PAR-4. PARs are widely distributed in the mammalian body, especially throughout the alimentary systems, and play various roles in physiological/pathophysiological conditions, i.e., modulation of salivary, gastric, or pancreatic glandular exocrine secretion, gastrointestinal smooth muscle motility, gastric mucosal cytoprotection, suppression/facilitation of visceral pain and inflammation, etc. Thus PARs are now considered novel therapeutic targets, and development of selective agonists and/or antagonists for PARs might provide a novel strategy for the treatment of various diseases that are resistant to current therapeutics.     

Activity of recombinant trypsin isoforms on human proteinase-activated receptors (PAR): mesotrypsin cannot activate epithelial PAR-1, -2, but weakly activates brain PAR-1

Trypsin-like serine proteinases trigger signal transduction pathways through proteolytic cleavage of proteinase-activated receptors (PARs) in many tissues. Three members, PAR-1, PAR-2 and PAR-4, are trypsin substrates, as trypsinolytic cleavage of the extracellular N terminus produces receptor activation. Here, the ability of the three human pancreatic trypsin isoforms (cationic trypsin, anionic trypsin and mesotrypsin (trypsin IV)) as recombinant proteins was tested on PARs.Using fura 2 [Ca(2+)](i) measurements, we analyzed three human epithelial cell lines, HBE (human bronchial epithelial), A549 (human pulmonary epithelial) and HEK (human embryonic kidney)-293 cells, which express functional PAR-1 and PAR-2. Human mesotrypsin failed to induce a PAR-mediated Ca(2+) response in human epithelial cells even at high concentrations. In addition, mesotrypsin did not affect the magnitude of PAR activation by subsequently added bovine trypsin. In HBE cells, which like A549 cells express high PAR-2 levels with negligible PAR-1 levels (<11%), half-maximal responses were seen for both cationic and anionic trypsins at about 5 nM. In the epithelial cells, mesotrypsin did not activate PAR-2 or PAR-1, whereas both anionic and cationic trypsins were comparable activators.We also investigated human astrocytoma 1321N1cells, which express PAR-1 and some PAR-3, but no PAR-2. High concentrations (>100 nM) of mesotrypsin produced a relatively weak Ca(2+) signal, apparently through PAR-1 activation. Half-maximal responses were observed at 60 nM mesotrypsin, and at 10-20 nM cationic and anionic trypsins.Using a desensitization assay with PAR-2-AP, we confirmed that both cationic and anionic trypsin isoforms cause [Ca(2+)](i) elevation in HBE cells mainly through PAR-2 activation. Desensitization of PAR-1 with thrombin receptor agonist peptide in 1321N1 cells demonstrated that all three recombinant trypsin isoforms act through PAR-1.Thus, the activity of human cationic and anionic trypsins on PARs was comparable to that of bovine pancreatic trypsin. Mesotrypsin (trypsin IV), in contrast to cationic and anionic trypsin, cannot activate or disable PARs in human epithelial cells, demonstrating that the receptors are no substrates for this isoenzyme. On the other hand, mesotrypsin activates PAR-1 in human astrocytoma cells. This might play a role in protection/degeneration or plasticity processes in the human brain.     

Dual modulation by thrombin of the motility of rat oesophageal muscularis mucosae via two distinct protease-activated receptors (PARs): a novel role for PAR-4 as opposed to PAR-1

Since protease-activated receptors (PARs) are distributed throughout the gastrointestinal tract, we investigated the role of PARs in modulation of the motility of the rat oesophageal muscularis mucosae. Thrombin produced contraction of segments of the upper and lower part of the smooth muscle. Trypsin contracted both the muscle preparations only at high concentrations. SFLLR-NH(2) and TFLLR-NH(2) (PAR-1-activating peptides), but not the PAR-1-inactive peptide FSLLR-NH(2), evoked a marked contraction. In contrast, the PAR-2 agonist SLIGRL-NH(2) and the PAR-4 agonist GYPGKF-NH(2) caused no or only a negligible contraction. In oesophageal preparations precontracted with carbachol, thrombin produced a dual action i.e. relaxation followed by contraction. TFLLR-NH(2) further contracted the precontracted preparations with no preceding relaxation. GYPGKF-NH(2), but not the inactive peptide GAPGKF-NH(2), produced marked relaxation. Trypsin or SLIGRL-NH(2) caused no relaxation. The PAR-1-mediated contraction was completely abolished in Ca(2+)-free medium and considerably attenuated by nifedipine (1 microM) and in a low Na(+) medium. The PAR-4-mediated relaxation was resistant to tetrodotoxin (10 microM), apamin (0.1 microM), charybdotoxin (0.1 microM), L-N(G)-nitroarginine methyl ester (100 microM), indomethacin (3 microM), propranolol (5 microM) or adenosine 3', 5'-cyclic monophosphorothioate, 8-bromo, Rp-isomer (30 microM). Thus, thrombin plays a dual role in modulating the motility of the oesophageal muscularis mucosae, producing contraction via PAR-1 and relaxation via PAR-4. The PAR-1-mediated effect appears to occur largely through increased Na(+) permeability followed by activation of L-type Ca(2+) channels and subsequent influx of extracellular Ca(2+). Our data could provide evidence for a novel role of PAR-4 as opposed to PAR-1, although the underlying mechanisms are still open to question.     

Ligand-specific roles for transmembrane 5 serine residues in the binding and efficacy of dopamine D(1) receptor catechol agonists

To refine further the structure-activity relationships of D(1) dopamine receptor agonists, we investigated the roles of three conserved serine residues [Ser198(5.42), Ser199(5.43), and Ser202(5.46)] in agonist binding and receptor activation. These transmembrane domain 5 (TM5) residues are believed to engage catechol ligands through polar interactions. We stably expressed wild-type or mutant (S198A, S199A, and S202A) D(1) receptors in human embryonic kidney cells. These receptors were expressed at similar levels (approximately 2000 fmol/mg) and bound the radioligand [(3)H]R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH 23390), although S198A and S199A displayed significant losses of affinity compared with that for wild-type receptors. The endogenous agonist, dopamine, had losses of potency at each of the mutant receptors. We tested cyclohexyl-substituted isochroman, carbocyclic, and chroman bicyclic dopamine analogs and found that the mutations affected the chroman to a lesser extent than the other compounds. These results support our hypothesis that the decreased D(1) activity of chroman analogs results from a ligand intramolecular hydrogen bond that impairs the ability of the catechol to engage the receptor. Sensitivities of these rigid catechol agonists to the effects of the serine mutations were dependent on ligand geometry, particularly with respect to the rotameric conformation of the ethylamine side chain and the distance between the amino group and each catechol hydroxyl. Functional experiments in striatal tissue suggest that the ability to engage TM5 serines is largely correlated with agonist efficacy for cAMP stimulation. These results provide a new understanding of the complexities of D(1) ligand recognition and agonist activation and have implications for the design of rigid catechol ligands.     

Impaired G protein coupling of the neurotensin receptor 1 by mutations in extracellular loop 3.

The neurotensin receptor 1, NTS1, is a G protein-coupled receptor. We have shown previously that the NTS1 receptor-binding site of the peptide agonist involved residues in extracellular loop 3 and at the extracellular junction of transmembrane domains 4 and 6. Here, we investigated by site-directed mutagenesis residues in extracellular loop 3 that might be involved in agonist-induced activation of the rat NTS1 (rNTS1) receptor. Wild type and mutated receptors were expressed in COS (African green monkey kidney fibroblasts) cells. Labeled agonist and antagonist binding as well as inositol phosphate and cAMP productions were studied. Compared to the wild type NTS1 receptor, the W339A, F344A, H348A and Y349A mutant receptors exhibited (i) decreased proportion of high over low affinity agonist binding sites, (ii) increased sensitivity of high affinity agonist binding to GTP gamma S, and (iii) impaired G protein coupling of high affinity agonist-receptor complexes. The data are consistent with the C-terminal part of extracellular loop 3 being essential for allowing high affinity agonist-NTS1 receptor complexes to couple to G proteins.