Detection of bradykinin B1 receptors in rat aortic smooth muscle cells.

The tritiated bradykinin B1 receptor agonist [3H]des-Arg(10)-kallidin bound to a single class of high-affinity binding sites (K(d) = 0.5 +/- 0.16 nM; B(max) = 15,000 +/- 8,000 sites/cell) on cultured rat aortic smooth muscle cells. [3H]Des-Arg(10)-kallidin association and dissociation kinetics were monoexponential, making it possible to determine the association and dissociation rate constants (k(+1) = 1.5 10(5) M(-1) sec(-1); k(-1) = 4.2 10(-5) sec(-1)). [3H]Des-Arg(10)-kallidin binding was inhibited by specific ligands of bradykinin B1 and B2 receptors with a rank order of potency consistent with that known for bradykinin B1 receptors in other species (des-Arg(9)-[Leu(8)]bradykinin = des-Arg(10)-kallidin = des-Arg(9)-bradykinin = des-Arg(10)-[Leu(9)]kallidin > des-Arg(10)-HOE-140 >> bradykinin >> HOE-140). Bradykinin B1 receptor mRNA was also detected in these cells. Des-Arg(10)-kallidin increased cytosolic free Ca2+ levels, phosphoinositide turnover, and arachidonic acid release at nanomolar concentrations (respective EC(50) values: 16 +/- 2, 4 +/- 2.7, 6 +/- 2 nM). These functional effects of des-Arg(10)-kallidin could be blocked by the bradykinin B1 receptor antagonist des-Arg(9)-[Leu(8)]bradykinin, but were not sensitive to bradykinin B2 receptor antagonists. These results therefore show that rat aortic smooth muscle cells in culture express functional bradykinin B1 receptors.

Characterisation of the effects of SR146131, a novel non-peptide CCK(1) receptor agonist, on IMR-32 human neuroblastoma cells.

The effect of ¿2-[4-(4-chloro-2, 5-dimethoxy-phenyl)-5-[2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoy l]-5, 7-dimethyl-indol-1-yl¿-acetic acid (SR146131), a novel non-peptide agonist of cholecystokinin (CCK) CCK(1) receptors, was compared to the effect of sulphated cholecystokinin octapeptide (CCK-8-S) on CCK(1) receptors of the human neuroblastoma cell line IMR-32. SR146131 inhibited [125I]CCK-8-S binding to IMR-32 cells at nanomolar concentrations. SR146131 and CCK-8-S increased intracellular free Ca(2+) levels ([Ca(2+)](i)) in the same concentration range (EC(50)=6+/-2.3 and 1.3+/-0.14 nM, respectively). Although the shape of the [Ca(2+)](i) increase induced by CCK-8-S and SR146131 was slightly different, extracellular Ca(2+) removal affected the response of both compounds to a similar degree, and the response of both compounds was essentially due to Ca(2+) release from intracellular stores. This was also confirmed by measuring the [Ca(2+)](i) response of single cells: both compounds induced [Ca(2+)](i) oscillations at subnanomolar concentrations and elicited a large peak increase in [Ca(2+)](i) at higher concentrations (EC(50)=0.5+/-0.04 and 5.7+/-1.9 nM for CCK-8-S and SR146131, respectively). Both CCK-8-S and SR146131 induced a sustained increase of phosphoinositide turnover in these cells, and acted at similar concentrations (EC(50)=2.7+/-0.7 and 6+/-3.1 nM, respectively), although the maximal effect of SR146131 was somewhat lower than the effect of CCK-8-S. These data show that SR146131 activates human CCK(1) receptors on IMR-32 cells in a manner and with a potency similar to that of CCK-8-S.

SR142948A is a potent antagonist of the cardiovascular effects of neurotensin.

The novel compound SR142948A was compared with SR48692 as an antagonist of neurotensin-induced cardiovascular effects both in vitro and in vivo. SR142948A inhibited [125I]-neurotensin binding [median inhibitory concentration (IC50) = 0.24 +/- 0.01 nM], neurotensin-induced cytosolic free Ca2+ increase (IC50 = 19 +/- 6 nM), and prostacyclin production in human umbilical vein endothelial cells (IC50 = 17 +/- 3 nM) at much lower concentrations than did SR48692 (respective IC50 values, 14 +/- 5, 41 +/- 16, and 86 +/- 16 nM). Oral administration of SR142948A (10 microg/kg) resulted in significant inhibition of neurotensin-induced blood pressure changes, whereas SR48692 was active only at 10-fold higher doses. Furthermore, SR142948A administered i.v. in microg/kg quantities in the rat was as active as mg/kg doses of SR48692 on neurotensin-induced increase in hematocrit. SR142948A injected intradermally also significantly inhibited neurotensin-induced plasma extravasation at concentrations as low as 10 pmol/site, whereas 1,000 pmol/site of SR48692 were necessary to reach a significant inhibition. These data show that SR142948A is a novel, extremely potent antagonist of neurotensin-induced cardiovascular responses both in vitro and in vivo. SR142948A and SR48692 constitute a pair of nonpeptide neurotensin antagonists of different potency, which may be used to probe for the implication of neurotensin receptors in physiologic or pathologic phenomena.

Neurotensin induces the release of prostacyclin from human umbilical vein endothelial cells in vitro and increases plasma prostacyclin levels in the rat.

Human umbilical vein endothelial cells express high affinity neurotensin receptors which are coupled to phosphoinositide turnover and 45Ca2+ efflux (Schaeffer et al., 1995. J. Biol. Chem. 270, 3409-3413). In order to assess the physiological significance of neurotensin receptor activation in endothelial cells, we have compared the in vitro effect of neurotensin on prostacyclin release and cytosolic free calcium increase ([Ca2+]i) as determined by fura-2 fluorescence experiments to the in vivo effect of neurotensin on blood pressure and haematocrit. Neurotensin increased [Ca2+]i levels at low concentrations (EC50 = 4.2 +/- 0.2 nM, n = 3). At similar concentrations, neurotensin was also able to induce prostacyclin release from human umbilical vein endothelial cells (EC50 = 14 +/- 1 nM, n = 3) as determined by a 6-keto-prostaglandin F1 alpha enzyme immunoassay. The neurotensin (100 nM)-induced [Ca2+]i increase and prostacyclin release were inhibited by the specific non-peptide neurotensin receptor antagonist SR 48692 at similar concentrations (IC50 = 41 +/- 16 nM and 86 +/- 17 nM, respectively, n = 3), confirming that these responses were mediated by high affinity neurotensin receptors. Intravenous injection of neurotensin (1-4 nmol/kg i.v.) in the rat resulted in a drop of blood pressure and increased haematocrit, and nearly doubled the plasma levels of 6-keto-prostaglandin F1 alpha, the stable metabolite of prostacyclin. Whereas indomethacin (10 mg/kg i.v.) pretreatment significantly reduced the effect of neurotensin on blood pressure, it did not alter its effect on haematocrit. These results suggest that prostacyclin release plays a role in the hypotensive effects of neurotensin, but is not involved in its effects on haematocrit.

Platelet-activating factor (PAF) is not an essential component of the cascade leading to smooth muscle cell proliferation following vascular injury.

In order to evaluate the relative importance platelet-activating factor (PAF) in the proliferative process leading to restenosis, the effect of SR 27417, a novel highly potent PAF receptor antagonist, on PAF-induced rabbit aortic smooth muscle cell (SMC) proliferation and intimal hyperplasia in rabbit carotid arteries subjected to air-drying endothelial injury was investigated. When added to low concentrations of foetal calf serum, PAF showed a dose-dependent mitogenic effect with regard to rabbit arterial SMC. SR 27417 inhibited PAF-induced SMC growth (IC50 = 2.4 +/- 0.4 nM) but remained without effect on the mitogenic effect of foetal calf serum. A 16 day treatment of SR 27417 (10 mg/kg per day, p.o.) abrogated PAF-induced platelet aggregation ex vivo but did not affect the development of intimal thickening, therefore showing that PAF is not an essential component of the cascade leading to restenosis following vascular injury.

Human umbilical vein endothelial cells express high affinity neurotensin receptors coupled to intracellular calcium release.

The binding of 125I-neurotensin (NT) to human umbilical vein endothelial cell monolayers was studied. At 20 degrees C, 125I-NT bound to a single class of binding sites with a dissociation constant of 0.23 +/- 0.08 nM and a binding site density of 5500 +/- 1300 sites/cell (n = 3). 125I-NT also bound to human aortic endothelial cells with a dissociation constant of 0.6 +/- 0.26 nM and a binding site density of 32000 +/- 1700 sites/cell. Association and dissociation kinetics were of a pseudo-first order and gave association and dissociation rate constant values of 1.6 x 10(6) M-1 s-1 and 3.5 x 10(-4) s-1, respectively. 125I-NT binding was inhibited by NT analogues with a rank order of potency similar to that characterizing brain high affinity NT binding sites (K0.5, nM): NT8-13 (0.11) > NT (0.35) > acetyl-NT8-13 (1.5) > [Phe11]NT (12) > [D-Tyr11]NT (> 1000). 125I-NT binding was also inhibited by the non-peptide NT antagonist SR 48692 (Ki = 16 nM) but was not affected by levocabastine, an inhibitor of low affinity brain NT binding sites. NT had no effect on cGMP levels in endothelial cells but NT and its analogues increased 45Ca2+ efflux from endothelial cells at nanomolar concentrations with a rank order of potency which was identical to that observed in binding experiments. This effect was inhibited by SR 48692 (IC50 = 8 nM). NT was able to increase phosphoinositide turnover in these cells, and this effect was blocked by SR 48692. The correlation between dissociation constants of NT analogues in binding experiments and IC50 values in 45Ca2+ efflux experiments was very high (r = 0.997) with a slope near unity, indicating that 125I-NT binding sites are functional NT receptors coupled to phosphoinositide hydrolysis and Ca2+ release in human umbilical vein endothelial cells.

Thrombin induces endothelial cell growth via both a proteolytic and a non-proteolytic pathway.

Binding of 125I-thrombin to human umbilical vein endothelial cells (HUVECs) was specifically displaced by the synthetic tetradecapeptide SFLLRNPNDKYEPF, named thrombin receptor agonist peptide (TRAP), which has recently been described as a peptide mimicking the new N-terminus created by cleavage of the thrombin receptor, and F-14, a tetradecapeptide representing residues 365-378 of the human alpha-thrombin B chain. Binding of 125I-TRAP to HUVECs was time-dependent, reversible and saturable, showing high affinity (KD = 1.5 +/- 0.4 microM) and high binding capacity (Bmax. = 7.1 +/- 0.6 x 10(6) sites/cell) (n = 3). Unlabelled thrombin and TRAP competitively and selectively inhibited the specific binding of 125I-TRAP with IC50 values of 5.8 +/- 0.7 nM and 2.8 +/- 0.4 microM respectively, whereas F-14 remained ineffective at displacing 125I-TRAP from its binding sites, suggesting the presence of at least two different types of thrombin-binding sites on HUVECs. TRAP was a potent mitogen for HUVECs in culture. Both TRAP and alpha-thrombin stimulated the proliferation of HUVECs with half-maximum mitogenic responses between 1 and 10 nM. F-14 also promoted HUVEC growth. The mitogenic effects of F-14 and TRAP were additive. N alpha-(2-Naphthylsulphonylglycyl)-DL-p-amidinophenylalanylpiper idine (NAPAP) and hirudin (two specific inhibitors of the enzyme activity of thrombin) specifically inhibited thrombin-induced HUVEC growth (IC50 values 400 +/- 60 and 52 +/- 8 nM respectively) but remained without effect on the mitogenic effect of TRAP or F-14. This demonstrated that the mitogenic effect of alpha-thrombin for HUVECs was intimately linked to its esterolytic activity but also showed that thrombin can stimulate HUVEC growth via another non-enzymic pathway. This hypothesis was further reinforced by the fact that F-14-induced proliferation of HUVECs remained unaltered by two antibodies directed against TRAP or the cleavage site on the extracellular portion of the thrombin receptor, which both strongly reduced thrombin-induced proliferation of HUVECs. Thrombin-, TRAP- or F-14-induced HUVEC proliferation was strongly inhibited by a neutralizing monoclonal antibody directed against basic fibroblast growth factor (bFGF), suggesting that thrombin regulates the autocrine release of bFGF in HUVECs.

Evidence for the existence of two different ADP-binding sites on rat platelets.

[3H]-2-Methylthio-ADP ([3H]-2-MeS-ADP), a stable analogue of ADP bound to one type of specific binding sites on rat platelets (KD = 0.77 +/- 0.07 nM, Bmax = 160 +/- 11 fmol/10(8) cells). 2-MeS-ADP and ADP antagonized [3H]-2-MeS-ADP binding, showing respective Ki values of 1.4 +/- 0.1 nM and 486 +/- 78 nM. Clopidogrel, a potent and specific inhibitor of ADP-induced platelet aggregation partially inhibited (approximately 70% inhibition) the binding of [3H]-2-MeS-ADP at the same time it abrogated 2-MeS-ADP- and ADP-induced adenylyl cyclase inhibition and aggregation. A population of clopidogrel-resistant [3H]-2-MeS-ADP binding sites was detected on platelets from treated animals. These receptor sites (KD = 0.9 +/- 0.2 nM, Bmax = 47 +/- 5 fmol/10(8) platelets) which showed high affinity for both ADP and 2-MeS-ADP (Ki values in the nanomolar range) might be involved in the ADP-induced shape change, a clopidogrel-resistant ADP-induced event. Using clopidogrel which acts via a direct and irreversible inhibition of ADP binding to its adenylyl cyclase-coupled receptor sites on platelets, we were able to discriminate between two types of ADP receptor sites. The former which was clopidogrel-sensitive represented about 70% of the total [3H]-2-MeS-ADP receptors and was responsible for ADP-induced platelet aggregation and adenylyl cyclase inhibition. The latter which was not affected by clopidogrel might be involved in ADP-induced shape-change.

Malformin-A1 inhibits the binding of interleukin-1 beta (IL1 beta) and suppresses the expression of tissue factor in human endothelial cells and monocytes.

Malformin-A1, a cyclic pentapeptide of microbial origin, antagonized in a competitive manner the binding of 125I-IL1 beta (interleukin-1 beta) to human monocytes and cultured human umbilical vein endothelial cells (HUVEC) with IC50 values (doses which reduce specific binding by 50%) of 250 +/- 80 and 230 +/- 25 nM, respectively (N = 3). IL1 increased in a dose-dependent manner the expression of tissue factor, a ubiquitous membrane-anchored glycoprotein that initiates blood coagulation at the surface of HUVEC and human monocytes. Malformin-A1 strongly inhibited IL1-induced tissue factor expression in HUVEC and monocytes with IC50 values of 420 +/- 35 and 105 +/- 25 nM, respectively (N = 3), and reduced IL1-induced expression of intercellular adhesion molecule-1 (ICAM-1, CD54) on HUVEC (IC50 = 125 +/- 18 nM) (N = 4). These observations demonstrate that malformin-A1 recognizes and blocks IL1 beta binding to its receptor sites on monocytes and endothelial cells and protects these cells from IL1-induced procoagulant changes.

Binding of [3H]-2-methylthio ADP to rat platelets--effect of clopidogrel and ticlopidine.

Thienopyridine compounds, including ticlopidine and clopidogrel, have been found to selectively inhibit adenosine 5' diphosphate (ADP)-induced platelet aggregation and adenylyl cyclase ex vivo, but the mechanism of their antiplatelet action remains to be determined. This study was aimed at investigating the effect of clopidogrel and ticlopidine on the binding of [3H]-2-methylthio- adenosine-5'-diphosphate (2-MeS-ADP) to rat platelets. Binding of [3H]-2-MeS-ADP to rat platelets was time-dependent and saturable. Scatchard analysis of the saturation binding data indicated that [3H]-2-MeS-ADP bound to one population of specific binding sites with high affinity (KD = 0.78 +/- 0.05 nM; Bmax = 156.3 +/- 4.8 fmole/10(8) cells) (n = 3). Unlabeled 2-MeS-ADP and ADP competitively and selectively inhibited the specific binding of [3H]-2-MeS-ADP with IC50 values of 11.3 +/- 1.2 nM and 11.3 +/- 0.7 microM, respectively (n = 3). Other nucleotide analogs such as ADP-beta S, ATP and ATP-alpha S also antagonized [3H]-2-MeS-ADP binding. When administered orally at doses ranging from 1 to 25 mg/kg, clopidogrel inhibited ADP- or 2-MeS-ADP-induced platelet aggregation as well as ADP or 2-MeS-ADP-induced inhibition of intraplatelet adenylyl cyclase. When measured in parallel, clopidogrel reduced in a dose-dependent manner the binding of [3H]-2-MeS-ADP to rat platelets ex vivo. Clopidogrel administration resulted in the decrease of [3H]-2-MeS-ADP binding sites on platelets without any significant change in the affinity; this indicates noncompetitive binding. Ticlopidine (200 mg/kg a day for 3 days) behaved in the same way.(ABSTRACT TRUNCATED AT 250 WORDS)

Chelerythrine, a selective protein kinase C inhibitor, counteracts pyrogen-induced expression of tissue factor without effect on thrombomodulin down-regulation in endothelial cells.

Endotoxin, interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) dose-dependently increased the expression of tissue factor and at the same time induced thrombomodulin down-regulation on the surface of cultured bovine aortic endothelial cells. Chelerythrine, a selective protein kinase C inhibitor, strongly reduced endotoxin-, IL1 beta- and TNF alpha-induced tissue factor expression but remained without effect with regard to thrombomodulin down-regulation measured in parallel. On the contrary, staurosporine, a highly potent, non-selective PKC inhibitor, simultaneously abolished tissue factor expression and thrombomodulin down-regulation induced by endotoxin, IL1 beta and TNF alpha. These results show that protein kinase C is deeply involved in the process leading to pyrogen-induced tissue factor expression and suggest that thrombomodulin down-regulation is regulated by a different pathway.

IL-4 and IL-13 exhibit comparable abilities to reduce pyrogen-induced expression of procoagulant activity in endothelial cells and monocytes.

Endotoxin (LPS), interleukin-1 beta (IL-1) and tumor necrosis factor-alpha (TNF) increased the expression of tissue factor, a membrane-anchored glycoprotein that initiates blood coagulation on the surface of cultured bovine aortic endothelial cells (ABAE) and human monocytes. These compounds simultaneously reduced the amount of thrombomodulin on the endothelial cell surface, further contributing to the procoagulant activity of the endothelium or monocytes. On endothelial cells and monocytes, interleukin-4 (IL-4) and interleukin-13 (IL-13), a newly described lymphokine, both strongly inhibited LPS-induced tissue factor expression, a similar activity also being obtained with regard to the pyrogenic effects of IL-1 or TNF. When measured in parallel, IL-4 and IL-13 counteracted thrombomodulin down-regulation induced by LPS, IL-1 or TNF in endothelial cells. These results therefore show that both IL-4 and IL-13 protect the endothelial and the monocyte surface against inflammatory mediator-induced procoagulant changes.

Biochemical and pharmacological properties of SR 27388, a dual antioxidant and PAF receptor antagonist.

SR 27388 (N-(2-dimethylaminoethyl)-N-(3-pyridinylmethyl[4-(3,5-di(tert- butyl)-4-hydroxylphenyl)thiazol-2-yl]amine) is a potent and competitive antagonist of the binding of [3H]PAF to its receptor on rabbit platelets exhibiting an equilibrium inhibition constant for PAF binding of 10.5 +/- 1.2 nM (n = 3). SR 27388 potently inhibited PAF-induced aggregation of rabbit platelets in vitro (IC50 = 65 +/- 12 nM) (n = 4). In this respect, SR 27388 was as potent as the triazolothienodiazepine WEB-2086 against PAF-induced aggregation of rabbit platelets and had no effect on the action of other platelet aggregating agents. SR 27388 prevented in a dose-dependent manner the formation of thiobarbituric acid reactive substances during membrane peroxidation (IC50 = 0.7 microM) and inhibited reduction of the stable 1,1-diphenyl-2-picrylhydrazyl radical, indicating that the antioxidant potency of SR 27388 was due to an efficient radical scavenging activity. SR 27388 displayed marked in vitro inhibition of zymosan-induced oxidative burst in human monuclear cells (IC50 = 3 microM). In vivo, SR 27388 protected mice from 100 micrograms/kg PAF-induced death with an ED50 value of 500 micrograms/kg, when given i.v., 5 min before PAF challenge or p.o. (ED50 = 800 micrograms/kg) when given 1 h before PAF administration. Similarly, i.v. or oral doses of SR 27388 afforded in mice complete protection against endotoxin-induced lethality (ED50 values were 250 micrograms/kg and 1.3 mg/kg, respectively). Neither BHT, vitamin E nor catechin exhibited significant protection against PAF- or endotoxin-induced death. In ovalbumin-presensitized rabbits, SR 27388 premixed with the allergen inhibited in a dose-dependent manner allergen-induced oedema formation in the skin (ED50 = 0.1 mumol/site). After an i.v. administration of 10 mg/kg, SR 27388 significantly protected mice against alloxan-induced diabetes. These results show that SR 27388 is a potent and orally active dual PAF receptor antagonist and antioxidant.

Effect of SR 27417 on the binding of [3H]PAF to rabbit and human platelets and human polymorphonuclear leukocytes.

SR 27417, the first member of a newly developed PAF antagonist series, fully and competitively displaced [3H]PAF from its high-affinity binding sites on washed rabbit and human platelets with Ki values of 57 +/- 0.02 and 50 +/- 0.8 pM (n = 3), respectively. Studies carried out in parallel demonstrated that SR 27417 was 5-7-times more potent than C16-PAF itself and more than 50-60-fold as active as the best synthetic PAF antagonist yet described. Additionally, SR 27417 selectively and competitively inhibited the specific binding of [3H]WEB-2086, a selective PAF receptor antagonist to its high-affinity receptors on washed rabbit platelets (IC50 = 0.18 +/- 0.01 nM). On human polymorphonuclear leukocytes, [3H]PAF bound to two classes of specific binding sites with high (KD = 0.31 +/- 0.05 nM; Bmax = 1.2 +/- 0.07 fmol/10(6) cells) and low affinity (KD = 11.1 +/- 1.5 nM; Bmax = 13.7 +/- 0.8 fmol/10(6) cells). On these cells, SR 27417 selectively inhibited the specific binding of [3H]PAF to its high- and low-affinity receptors (IC50 values of 0.17 +/- 0.02 and 6.9 +/- 0.7 nM, respectively) and displayed the same inhibitory pattern as that already reported on platelets. In conclusion, SR 27417 can be considered as the most potent PAF receptor antagonist described to date.

Binding of [3H]SR 27417, a novel platelet-activating factor (PAF) receptor antagonist, to rabbit and human platelets and polymorphonuclear leukocytes.

Binding of [3H]SR 27417 to washed rabbit platelets was time-dependent and saturable. [3H]-SR 27417 binding was reversible after short incubation periods but became progressively irreversible when incubated for more than 2 hr. Scatchard analysis of the saturation binding data indicated that [3H]-SR 27417 bound to two populations of specific binding sites with high (KD = 0.75 +/- 0.06 nM; Bmax = 58.7 +/- 4.3 fmol/10(8) cells; N = 3) and low affinity (KD = 53.8 +/- 4.9 nM; Bmax = 1665 +/- 87 fmol/10(8) cells; N = 3). Unlabelled C16-PAF competitively and selectively inhibited the specific binding of [3H]SR 27417 with an IC50 value of 1.9 +/- 0.04 nM (N = 3). SR 27414 fully and competitively displaced [3H]SR 27417 from its binding sites on rabbit platelets with a Ki value of 260 +/- 20 pM (N = 3) therefore demonstrating that SR 27417 was more potent than C16-PAF itself. On washed human platelets, [3H]SR 27417 displayed specific as well as saturable binding to two populations of binding sites (KD = 0.21 +/- 0.01 nM; Bmax = 13.9 +/- 0.9 fmol/10(8) cells and KD = 4.75 +/- 1.9 nM; Bmax = 82.2 +/- 2.9 fmol/10(8) cells; N = 3) for high- and low-affinity binding sites, respectively, whereas [3H]SR 27417 bound to only one single class of binding sites on human polymorphonuclear leukocytes (KD = 0.31 +/- 0.1 nM; Bmax = 9.36 +/- 1.2 fmol/10(6) cells; N = 3). IC50 values for C16-PAF, SR 27417 and other PAF receptor antagonists on all three cell types indicated that SR 27417 was at least three times more potent than C16-PAF itself and more than 30-fold as active as the best synthetic PAF receptor antagonist tested (L 659,989). In conclusion, these data indicate that SR 27417 appears to be one of the most potent PAF receptor antagonists yet described, as well as a suitable radioligand for labelling PAF receptors on intact blood cells.

The NK1 receptor is involved in the neurokinin-induced shape change of rabbit platelets.

Substance P and selective neurokinin receptor agonists have been tested for their ability to induce shape change in rabbit platelets. Substance P and the NK1 receptor agonist Ac [Arg6,Sar9,Met(O2)11]-substance P (6-11) induced shape change (EC50 = 3 and 6 nM, respectively), whereas the selective NK2 agonist [Nle10]-Neurokinin A (4-10) and the selective NK3 agonist [MePhe7]-Neurokinin B did not show any effect. Moreover, the specific NK1 receptor antagonist CP-96,345 selectively and dose-dependently counteracted the effect of substance P or of the NK1 receptor agonist (IC50 = 2 and 0.8 nM, respectively), whereas the selective NK2 receptor antagonist, SR 48968, had no effect. Unlike for serotonin or low doses of ADP, epinephrine did not allow substance P or the NK1 receptor agonist to become a proaggregating substance. These data therefore show that the NK1 receptor is solely involved in the neurokinin-induced shape change of rabbit platelets.

IL-4 inhibits LPS-, IL-1 beta- and TNF alpha-induced expression of tissue factor in endothelial cells and monocytes.

Inflammatory mediators such as endotoxin, interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) dose-dependently increased the expression of tissue factor on the surface of cultured bovine aortic endothelial cells (ABAE), human umbilical vein endothelial cells (HUVEC) and human monocytes. In ABAE, endotoxin-, IL-1 beta- and TNF alpha-induced tissue factor expression was suppressed by interleukin-4 (IL-4) which also neutralized the pyrogenic effect of endotoxin in HUVEC and monocytes. IL-4 did not alter TNF-alpha-induced procoagulant changes in HUVEC and monocytes but strongly protected the monocyte surface against IL-1 beta-induced procoagulant changes.

Effect of SR 27417 on oedema formation induced in rabbit skin by platelet-activating factor or antigen.

SR 27417, the first member of a newly developed series of platelet activating factor (PAF) antagonists inhibited in a dose-dependent manner PAF-induced oedema formation in rabbit skin when administered i.d. premixed with PAF (ED50 = 3.3 +/- 0.15 pmol/site i.d. (intradermally) (n = 5). The effect of SR 27417 was over 660 times more potent than that of the triazolothienodiazepine, WEB-2086 (ED50 = 5.4 +/- 0.71 nmol/site i.d.) (n = 5). SR 27417 protected rabbits from PAF-induced plasma extravasation with an ED50 value of 16 +/- 3 micrograms/kg when given i.v. 1 h before PAF challenge. It was also effective when given p.o. 3 h before PAF i.d. administration (ED50 = 0.18 +/- 0.07 mg/kg p.o.) (n = 5). This effect of SR 27417 was selective for PAF since inflammatory responses induced by other mediators (bradykinin, histamine, N-formyl-L-methionyl-L-leucyl-L-phenyl-alanine, leukotriene B4) were not affected. After i.v. or oral administration (1 and 5 mg/kg respectively) SR 27417 had an extended duration of activity (between 72 and 96 h). In presensitized rabbits, SR 27417 premixed with the allergen inhibited dose-dependently allergen-induced plasma exudation (ED50 = 12 +/- 0.08 nmol/site i.d.) (n = 5) (vs. 850 +/- 98 nmol/site (n = 5) for WEB-2086). Similarly, i.v. injection of SR 27417 (1 mg/kg i.v.) inhibited allergen-induced vascular permeability. These results confirm that PAF plays a major role in the development of cutaneous anaphylaxis and that SR 27417 may be an effective prophylactic drug.

Ex vivo effects of SR 27417, a novel PAF antagonist, on rabbit platelet aggregation and [3H]-PAF binding.

Pharmacodynamics of SR 27417, a novel, specific platelet-activating factor (PAF) antagonist was monitored with ex vivo PAF-induced platelet aggregation in the rabbit. Single per os administration of SR 27417 (5 mg/kg) resulted in complete inhibition of this aggregation for at least 3 days. The magnitude of this inhibitory effect was dose-dependent with a maximum effect 1-3 h after oral administration. IC50 values for PAF-induced platelet aggregation were 80, 35, 50 and 1250 micrograms/kg, 1, 3, 24 and 72 h after oral intake of SR 27417 respectively. This effect was irreversible in nature since it could not be overcome by prolonged incubation of platelets isolated from treated animals in plasma from controls or in buffer. Examination of [3H]-PAF binding to washed platelets isolated 5 min after i.v. administration of increasing concentrations of SR 27417 revealed a competitive-type of inhibition whereas 24 h after p.o. administration, SR 27417 antagonized [3H]-PAF binding in a non-competitive manner.

Biochemical and pharmacological activities of SR 27417, a highly potent, long-acting platelet-activating factor receptor antagonist.

SR 27417 [N-(2-dimethylamino ethyl)-N-(3-pyridinyl methyl)[4- (2,4,6-triisopropylphenyl) thiazol-2-yl]amine] is the first member of a newly developed platelet-activating factor (PAF) antagonist series. It is a highly potent, competitive and selective antagonist of the binding of [3H]PAF to its receptor in rabbit platelets. It exhibits an equilibrium inhibition constant for PAF binding of 57 pM, a value that is at least 5-fold lower than that of unlabeled PAF itself. SR 27417 potently inhibited PAF-induced aggregation of rabbit and human platelets in vitro (IC50 = 0.10 and 0.50 nM, respectively) but had no effect on the action of other platelet-aggregating agents. In comparison with the triazolothienodiazepine WEB-2086, SR 27417 was 470 and 70 times more potent against PAF-induced aggregation of rabbit and human platelets, respectively. SR 27417 displayed marked in vitro inhibition of PAF-induced oxidative burst in guinea pig macrophages (IC50 = 32 nM). In an in vivo model, it protected mice from 100 micrograms/kg PAF-induced death when given i.v. (ED50 = 7.5 micrograms/kg) 5 min before PAF challenge or p.o. (ED50 = 45 micrograms/kg) 3 hr before PAF administration. SR 27417 inhibited PAF-induced death in mice with an impressive p.o. or i.v. duration of action of 30 to 48 hr. In anesthetized guinea pigs, SR 27417 inhibited i.v. and p.o. 100 ng/kg PAF-induced bronchoconstriction (ED50 = 14 and 140 micrograms/kg, respectively), hemoconcentration (ED50 = 20 and 270 micrograms/kg, respectively), thrombocytopenia (ED50 = 30 and 240 micrograms/kg, respectively) and leukopenia (ED50 = 0.1 and 1.5 mg/kg, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)