Tritiated photoactivatable analogs of the native human thrombin receptor (PAR-1) agonist peptide, SFLLRN-NH2.

Six photoactivatable analogs of the human thrombin receptor activating peptide (TRAP), SFLLRN-NH2, were synthesized by substituting the photoactive amino acid, p-benzoylphenylalanine (Bpa), into each position of the peptide sequence. Platelet aggregation assays indicated that the peptides with Bpa substitutions at positions 3 to 6 retained agonist activity. These peptides were prepared in tritiated form as potential thrombin receptor photoaffinity labels. The [3H]Bpa-containing analogs were constructed by resynthesizing the peptides with the amino acid, 4-benzoyl-2',5'-dibromophenylalanine (Br2Bpa), and subjecting the purified peptides to Pd-catalyzed tritiodebromination. The radiochemical yields for the reductive tritiation were < 2% for peptides with [3H]Bpa in the third and fourth positions, and between 7 and 16% for the peptides with substitutions at the fifth and sixth positions. The low yields were due to over-reduction of the Bpa carbonyl group and nonspecific degradation during reductive tritiation. This report describes the first use of Br2Bpa for the preparation of tritiated photoactivatable peptides.     

PAR-3 is a low-affinity substrate,high affinity effector of thrombin

A polypeptide corresponding to the extracellular domain of protease-activated receptor 3 (PAR-3) is hydrolyzed by thrombin slowly because of high K(M) (>100 microM). However, thrombin is found to bind two PAR-3, one without catalyzing hydrolysis or blocking the active site, while the other is hydrolyzed. In a solvent lacking Na(+), hydrolysis of a nitroanilide substrate is enhanced 1.6-fold by addition of PAR-3 polypeptide, with half-saturation at 2.5 microM. In contrast, the fibrinogen clotting activity of thrombin is inhibited completely by PAR-3, with a K(I) of 3 microM. None of the activities of thrombin are affected by addition of 50 microM PAR-4 polypeptide. Thus, PAR-3 in low concentrations binds thrombin in a configuration that blocks the anion-binding exosite but not the catalytic site, while hydrolysis of PAR-3, PAR-4, and other substrates that do not interact with exosite I persists. The allosteric effect of PAR-3 is characteristic of that of Na(+).     

Discovery and synthesis of a novel series of quinoline-based thrombin receptor (PAR-1) antagonists

The design, synthesis, and SAR studies of a structurally novel series of highly potent thrombin receptor (PAR-1) antagonists are described. Compound 30 is a highly potent thrombin receptor antagonist (IC(50)=6.3 nM), a related compound 36 showing efficacy in a monkey ex vivo study.     

High-affinity thrombin receptor (PAR-1) ligands: a new generation of indole-based peptide mimetic antagonists with a basic amine at the C-terminus

A new generation of indole-based peptide mimetics, bearing a basic amine at the C-terminus, was developed by the agency of two complementary, multistep, trityl resin-based approaches. Thus, we obtained several high-affinity thrombin receptor (PAR-1) ligands, such as 32 and 34. Compounds 32 and 34 were found to bind to PAR-1 with excellent affinity (IC(50)=25 and 35 nM, respectively) and to effectively block platelet aggregation induced by SFLLRN-NH(2) (TRAP-6) and alpha-thrombin.     

Use of a novel peptide leukotriene receptor antagonist, Ly-163443, in splanchnic artery occlusion shock

We studied the effects of LY-163443, a novel selective receptor antagonist of LTD4 and LTE4, in splanchic artery occlusion (SAO) shock. LY-163443 antagonized the bronchoconstrictor effect of LTD4 given intravenously to anesthetized rats. Anesthetized rats subjected to total occlusion of the superior mesenteric and the celiac arteries for 40 minutes developed a severe shock state usually resulting in a fatal outcome within two hours after release of the occlusion. SAO shock rats pre-treated with LY-163443 before the occlusion of the splanchnic arteries maintained post-release MABP at significantly higher values compared to rats receiving either the vehicle or LY-163443 as a post-treatment 15 min after occlusion (final MABP 96 +/- 8 vs 51 +/- 1, p less than 0.01 and 53 +/- 3, p less than 0.01, respectively). Pre-treatment with LY-163443 attenuated the release of the lysosomal hydrolase, cathepsin D (p less than 0.01 from vehicle and p less than 0.05 from post-treatment groups), and the plasma accumulation of free amino-nitrogen compounds (p less than 0.05 from vehicle). Furthermore, the plasma activity of a myocardial depressant factor (MDF) was significantly lower in the pre-treatment group than in the vehicle group (27 +/- 3 vs 51 +/- 6 U/ml, p less than 0.01). SAO shock rats pretreated with LY-163443 also exhibited significantly higher survival rates (p less than 0.01 from vehicle and post-treatment groups), and prolonged survival times (p less than 0.01 from vehicle and post-treatment groups).(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical synthesis, purification, and characterization of two inflammatory proteins, neutrophil activating peptide 1 (interleukin-8) and neutrophil activating peptide

Two recently identified pro-inflammatory proteins, namely, neutrophil activating peptide 1 (NAP-1) [also termed interleukin-8 (IL-8)] and NAP-2, were chemically synthesized, purified, and characterized. The fully protected NAP-1/IL-8 (72 residues) and NAP-2 (70 residues) peptide chains were assembled by automated solid-phase methods with average stepwise yields of 99.5 and 99.3%, resulting in overall chain assembly yields of 70 and 62%, respectively. Deprotection resulted in crude products, which were allowed to fold by air oxidation, and were purified by two cycles of reverse-phase high-pressure liquid chromatography, yielding 27 mg of NAP-1/IL-8 and 22 mg of NAP-2. Purity was established by reverse-phase high-pressure liquid chromatography and isoelectric focusing, and the primary structures of the purified products were verified by using mass spectrometry and Edman sequencing methods. Synthetic and recombinant NAP-1/IL-8 were equally active on human neutrophil granulocytes as determined by measuring the induction of cytosolic free calcium, elastase release, and chemotaxis. Synthetic NAP-2 was equivalent to purified natural NAP-2 in the elastase release and calcium mobilization assays, but it was consistently less potent (3-5-fold) as a stimulus of chemotaxis, perhaps indicative of additional chemotactic components in the natural preparation. The results indicate that by chemical synthesis these cytokines can be obtained in purity and quantities suitable for further structural analysis, as well as functional studies both in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of calcium on in vitro contractility of human umbilical artery

The action of calcium on human umbilical artery was studied by an in vitro perfusion method. An increase in tonus and intense spontaneous activity were observed in the preparations at high calcium concentrations. A marked relaxation of the umbilical artery occurred in the absence of calcium. Estriol had a relaxing effect on the umbilical vessels which was more intense at calcium concentrations near physiological levels. Estriol also abolished spontaneous contractions. In the absence of calcium, estriol showed no relaxing effect. The results suggest a participation of calcium in the mechanism of action of estriol on human umbilical artery in vitro.     

A novel hypothalamic peptide, pituitary adenylate cyclase activating peptide, modulates Sertoli cell function in vitro.

Pituitary adenylate cyclase-activating peptide (PACAP), a novel hypothalamic peptide that has been shown to exist in several tissues including the testis, was examined for its effects on cultured rat Sertoli cells. PACAP stimulates cAMP accumulation in Sertoli cells cultured from 15-day-old rats in the presence or absence of methylisobutylxanthine, a phosphodiesterase inhibitor, and in the presence of pertussis toxin, a blocker of the adenylate cyclase inhibitory pathway. Maximal stimulation, which is 20-40% of that attainable with FSH, occurs at PACAP concentrations of 10 nM: the ED50 is approximately 100 pM. The ability of PACAP to stimulate Sertoli cell cAMP declines with increasing age of donor animals (15-60 days of age) in a fashion similar to the FSH effect. PACAP stimulation of Sertoli cell cAMP accumulation is additive with submaximal, but not maximal, concentrations of FSH or forskolin. PACAP also stimulates the secretion of lactate, estradiol, and inhibin in a concentration-dependent manner. The stimulation of Sertoli cell cAMP accumulation by PACAP is not altered by a vasoactive intestinal peptide antagonist, and vasoactive intestinal peptide alone does not stimulate cAMP accumulation, indicating that PACAP is not acting via vasoactive intestinal peptide receptors. Further experiments are needed to determine whether PACAP is synthesized within the testis and if so, in which cell types; however, the present data clearly demonstrate that PACAP can modulate Sertoli cell function in vitro.     

Human recombinant lipocortin 1 inhibits prostacyclin production by human umbilical artery in vitro

Submicromolar concentrations of human recombinant Lipocortin 1 inhibit the release of prostacyclin from human umbilical artery rings in a dose-dependent fashion. This is the first demonstration that the recombinant protein is effective in human cells.     

The effect of selective umbilical embolization on the common umbilical artery pulsatility index and umbilical vascular resistance in fetal sheep.

In eight anaesthesized fetal sheep (gestational age 112-127 days; term 147 days), embolization of the umbilical placental circulation was performed in order to evaluate the response of the umbilical artery pulsatility index to an exclusive increase in umbilical vascular resistance. Measurements were performed using a 20 MHz pulsed Doppler transducer and an electromagnetic flow meter mounted on the common umbilical artery and catheters at the aortic trifurcation and in one of the umbilical veins. Umbilical vascular resistance was calculated according the Poiseuille equation as the ratio of aortic to umbilical venous pressure gradient and umbilical blood flow. Microspheres were administered at 15-min intervals through a catheter in one of the cotyledonary arteries, until fetal heart rate had decreased beneath 100 beats/min or had become arrhythmic. The period of examination per fetus varied between 60 and 120 min, after which cardiac decompensation occurred. During this period, umbilical perfusion pressure increased from 20.3 +/- 4.9 to 28.1 +/- 4.7 mmHg (SD; P less than 0.01), umbilical blood flow (ml/min) decreased from 342 +/- 127 to 115 +/- 99 mmHg (SD; P less than 0.01), umbilical vascular resistance increased from 0.065 +/- 0.022 to 0.342 +/- 0.150 mmHg.min/ml (P less than 0.01) and common umbilical artery pulsatility index increased from 0.97 +/- 0.23 to 4.03 +/- 1.69 (P less than 0.01). Fetal heart rate did not change significantly (168 +/- 33 prior to cardiac decompensation versus 178 +/- 19 beats/min at baseline condition). The linear correlation between common umbilical artery pulsatility index and umbilical vascular resistance varied between 0.83 and 0.99 and the average correlation was 0.93 (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of substance P, calcitonin gene-related peptide and capsaicin on tension and membrane potential of pig coronary artery in vitro

The effects of substance P (SP), calcitonin gene-related peptide (CGRP) and capsaicin were studied on isometric tension and membrane potential of pig coronary arterial strips in vitro. CGRP induced an endothelium-independent relaxation without change in the smooth muscle membrane potential whereas SP relaxed and hyperpolarized the strip via the endothelium. Applied together, the mechanical effects of SP plus CGRP were additive. CGRP did not affect the hyperpolarization due to SP. In order to examine a possible role of endogenous SP and CGRP, capsaicin was used. It provoked a contraction which was adventitia-dependent, and was inhibited by indomethacin. In presence of indomethacin, capsaicin caused a relaxation. It was accompanied by a hyperpolarization of smooth muscle membrane potential only when the strip had an intact endothelium. When the strip was de-endothelialized capsaicin relaxation subsisted. This indicates that capsaicin produced a relaxation of indomethacin-treated strip by releasing a hyperpolarizing endothelial factor and probably endogenous CGRP.     

Discovery and initial structure-activity relationships of trisubstituted ureas as thrombin receptor (PAR-1) antagonists

Thrombin is the most potent agonist of platelet activation, and its effects are predominantly mediated by platelet thrombin receptors. Therefore, antagonists of the thrombin receptor have potential utility for the treatment of thrombotic disorders. Screening of combinatorial libraries revealed 2 to be a potent antagonist of the thrombin receptor. Modifications of this structure produced 11k, which inhibits thrombin receptor stimulated secretion and aggregation of platelets.     

Mutation of W215 compromises thrombin cleavage of fibrinogen, but not of PAR-1 or protein C

W215 is a highly conserved residue that shapes the S3 and S4 specificity sites of thrombin and participates in an edge-to-face interaction with residue F8 of the fibrinogen Aalpha chain. Protein C and the platelet receptor PAR-1 carry an acidic residue at P3 and bind to the active site of thrombin without making contact with W215. This suggested that mutation of W215 could dissociate the cleavage of fibrinogen from that of protein C and PAR-1. Replacement of W215 with Phe produces modest effects on thrombin function, whereas the W215Y replacement compromises significantly the catalytic activity toward all chromogenic and natural substrates that are tested. Replacement of W215 with Ala almost obliterates Na(+) binding, reduces the level of fibrinogen cleavage 500-fold, but decreases the levels of protein C activation and PAR-1 cleavage only 3- and 25-fold, respectively. The W215A mutant cleaves PAR-1 with a specificity constant that is more than 13-fold higher than that of fibrinogen and protein C and is the first thrombin derivative to be described that functions as an almost exclusive activator of PAR-1. The environment of W215 influences differentially three physiologically important interactions of thrombin, which should assist in the study of each of these functions separately in vivo.     

Effects of the calcium antagonist, diltiazem on in vitro and in vivo/in vitro bone resorption

The effect of the calcium antagonist diltiazem on bone resorption in organ culture has been investigated. It was found that diltiazem was ineffective alone but that in concentrations above 5 mumol/l it reduced mineral and organic resorption induced in vitro by 1.25 dihydroxycholecalciferol (1.25 (OH)2D3). No additivity with calcitonin effects was observed. Diltiazem did not significantly affect bone resorbing activity stimulated by 24,25(OH)2D3. Bone resorption was measured by an in vivo/in vitro technique using 45Ca prelabelled mice. Compared with 1.25(OH)2D3 alone treated group (0.480 pmol/g), it was found that diltiazem (100 nmol/g) reduced bone resorption without effect on calcium and phosphorus plasmatic concentrations at death. These data suggest that such a calcium antagonist is able to inhibit 1.25-(OH)2D3-increased-bone resorption either in vitro or in vivo/in vitro.     

Thrombin and PAR-1 acitvating peptide increase iNOS expression in cytokine-stimulated C6 glioma cells

Thrombin (THR) plays a key role in the brain under physiological and pathological conditions. Several of the biological activities of thrombin have been shown to be mainly driven through activation of protease-activated receptor-1 (PAR-1)-type thrombin receptor. Here we have studied the effect of THR and PAR-1-activating peptide (PAR1-AP), SFLLRN, on cytokine-induced expression of inducible nitric oxide (iNOS), a prominent marker of astroglial activation using the rat C6 glioma cells. In this cell line, THR (1-10 U/mL) and PAR1-AP (1-100 microM) induced a significant concentration-dependent increase both of IFN-gamma- (250 U/mL) or TNF-alpha- (500 U/mL) induced NO release. The observed increase of NO production was related to an enhancement of iNOS expression as measured in cell lysates prepared from different treatments by using SDS-PAGE followed by western blot analysis. The effect of THR, but not that of PAR1-AP, was significantly inhibited by hirulog(TM) (60 microg/mL), a specific and stochiometric THR inhibitor or by cathepsin-G (40 mU/mL), an inhibitor of PAR-1. In conclusion our data suggest a role for THR through activation of PAR-1 in the induction of astroglial iNOS, and further support the hypothesis that THR may function as an important pathophysiological modulator of the inflammatory response.     

Synergistic actions of a thrombin-derived synthetic peptide and a thrombin receptor-activating peptide in stimulating fibroblast mitogenesis

We measured the ability of the thrombin receptor activating peptide, SFLLR-NH₂ (P5A) to stimulate ³H-thymidine incorporation in hamster CCL-39 fibroblasts either alone or in combination with the thrombin-derived polypeptides, YPPWNKNFTENDLL (TDP-1) and AGYKPDEGKRGDACEGDSGGPFV (TDP-2). In the presence (but not absence) of the amino peptidase inhibitor amastatin (10 μM), P5A alone (7.5 to 100 μM) caused a 1.5- to 2-fold stimulation of thymidine incorporation above basal, even though this inhibitor did not abrogate the degradation of P5A by other peptidases present in the assay medium. Neither TDP-1 nor TDP-2 alone had any effect on thymidine incorporation. However, TDP-1 (30 to 90 μM) considerably augmented P5A-mediated thymidine incorporation at low P5A concentrations (7.5 to 30 μM), shifting the P5A concentration-effect curve to the left. TDP-2 was inactive in this regard. The EC₅₀ for this potentiating action of TDP-1 was approximately 40 μM. Further, thrombin, rendered proteolytically inactive by a low-molecular-weight bifunctional inhibitor, hirutonin-6, also acted synergistically with P5A to stimulate CCL-39 cell thymidine incorporation. We hypothesize that thrombin can cause its cellular effects, such as thymidine incorporation, not only via the proteolytic activation of its G-protein-coupled receptor, but also via the concurrent and synergistic interaction of its TDP-1 peptide domain with a separate cell surface docking site. © 1996 Wiley-Liss, Inc.     

Effects of 3,4-dihydroxyacetophenone on the biosynthesis of TXA2 and PGI2 in human placental villus and umbilical artery segments in vitro

In this paper, the effects of 3,4-dihydroxyacetophenone, DHAP (Qingxintong), an active constituent of traditional Chinese medicine, on the biosynthesis of TXA2 and PGI2 in human placental villi and umbilical artery segments of normal term pregnancy in vitro were studied by a perifusion technique. The collected fractions were assayed by radioimmunoassay for TXB2 and 6-keto-PGF1 alpha. The results showed that DHAP inhibited TXA2 and PGI2 production by umbilical artery segments in a dose dependent fashion and in both tissues TXA2 was more sensitive to inhibition than was 6-keto-PGF1 alpha. According to these data it is suggested that DHAP might be useful in treatment of pathologic pregnancies with chronic defective utero-placental circulation such as PIH and IUGR to improve this circulation.     

Jab1, a novel protease-activated receptor-2 (PAR-2)-interacting protein, is involved in PAR-2-induced activation of activator protein-1

Protease-activated receptor-2 (PAR-2), a G protein-coupled receptor for trypsin and tryptase, exerts important physiological and pathological functions in multiple systems. However, unlike PAR-1, the PAR-2-mediated intracellular signal transductions are hardly known. Here, using yeast two-hybrid screening with a human brain cDNA library, we identified an interacting partner of human PAR-2, the Jun activation domain-binding protein 1 (Jab1). The interaction was confirmed by glutathione S-transferase pull-down assays in vitro, and by co-immunoprecipitation assays in vivo. Jab1 was also shown to be colocalized with PAR-2 in both transfected HEK293 cells and in normal primary human astrocytes by double immunofluorescence staining. Further experiments demonstrated that multiple intracellular domains of PAR-2 are required for the interaction with Jab1. We then showed that agonist stimulation of PAR-2 disrupted the interaction, which could be prevented by the inhibitor of receptor endocytosis phenylarsine oxide, but not by the lysosomal protease inhibitor ZPAD. Importantly, we found that activation of PAR-2 induced the redistribution of Jab1 from the plasma membrane to the cytosol, but did not influence expression of Jab1. Furthermore, Jab1 mediated PAR-2-induced c-Jun activation, which was followed by increased activation of activator protein-1. Loss-of-function studies, using Jab1 small interfering RNA, demonstrated that Jab1 knockdown blocked PAR-2-induced activator protein-1 activation. Taken together, our data demonstrate that Jab1 is an important effector that mediates a novel signal transduction pathway for PAR-2-dependent gene expression.     

Effect of a novel vasoconstrictor endothelin-1 (1-31) on human umbilical artery

To clarify the action of a novel endothelin-1 with 31 amino acids, ET-1 (1-31), on fetal circulation, its vasoconstrictive activity on human umbilical and uterine arteries was investigated in comparison with that of a conventional ET-1 (1-21). UFER micro-easy magnus was used for determination of vasoconstriction. The contraction of umbilical artery by KCl was significantly weaker than that of the uterine artery. In ETs, constriction by KCl was set as control, and the rate of constriction of uterine and umbilical arteries was used for comparison. The constriction of human uterine artery induced by ET-1 (1-31) was also significantly weaker than that by ET-1 (1-21). On the contrary, ET-1 (1-31) was a potent constrictor on the umbilical artery equally to ET-1 (1-21). The present study is the first to demonstrate that ET-1 (1-31) has a contractile activity on human vessels. Furthermore, the regulatory mechanism on constriction of umbilical artery is different from that observed in a systemic vessel, indicating a particularly important role of ET-1 (1-31) in fetal circulation.     

Effects of HA-1077 and Y-27632, two rho-kinase inhibitors, in the human umbilical artery

A role for the small G protein rho and rho-kinase has been shown in smooth muscle contraction regarding Ca⁺⁺ sensitivity. However, there are no data in the literature assessing how this system operates in human umbilical arteries (HUA). Therefore, we evaluated the effects of HA-1077 and Y-27632, two rho-kinase inhibitors, on agonist-(5-hydroxytryptamine [5-HT]) and depolarization-induced (KCl) contractions of HUA. HA-1077 and Y-27632 inhibited 5-HT-induced contractile responses at 10⁻⁴ M concentration but not at 10⁻⁵ M. HA-1077 at 10⁻⁴ M also significantly attenuated contractions induced by 20 mM KCl. In addition, HUA precontracted with 5-HT relaxed concentration dependently in response to HA-1077 and Y-27632. When precontracted with KCl, HUA also relaxed dose-dependently in response to HA-1077, but the maximal relaxation was significantly smaller than the response obtained when precontracted with 5-HT. To determine possible involvement of rho-kinase on agonist-induced intracellular calcium-mediated contractions, tissues were precontracted with 5-HT in Ca⁺⁺-free Krebs solution before cumulative addition of HA-1077 or Y-27632 (10⁻⁷ to 10⁻⁴ M). Both rho-kinase inhibitors relaxed HUA completely. Maximum relaxations of HUA to HA-1077 and Y-27632 were significantly larger than the responses seen in normal Krebs solution and were obtained with lower concentrations of the drugs considered to be more specific for rho-kinase inhibition. However, preincubation of HUA with HA-1077 or Y-27632 (10⁻⁵ M for both) did not affect the 5-HT-induced contractions in this medium. Finally, immunoblot experiments revealed the expression of rho-kinase isoform rockII protein in HUA. These results indicate that rhoA/rho-kinase pathway can contribute to agonist-induced contractions of HUA. However, this effect appears to be limited to intracellular calcium-induced contractions and may be more important in sustaining contractions rather than the initial phase of force development.