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Biomedical subjects

J M Herbert

Publications and source records attributed to J M Herbert.

At least 19 recordsLinked to original sources
PubMed1992

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.

Animals↗
PubMed1992

Importance of platelets in experimental venous thrombosis in the rat.

Venous thrombosis was induced by ligature of the inferior vena cava in rats whose blood was made hypercoagulable by intravenous (IV) administration of tissue thromboplastin. From a dose-response showing that the administration of increasing doses of tissue thromboplastin resulted in a subsequent progressive increase of thrombus weight, two concentrations of tissue thromboplastin were chosen: a high dose (550 microL/kg, IV) where thrombus formation was optimal and a concentration (7 microL/kg, IV) where tissue thromboplastin-hypercoagulability was intermediate. In both experimental conditions, leukopenia provoked by a myelotoxic drug did not influence the development of venous thrombosis. However, after thrombocytopenia induced by an antiplatelet antiserum, a dramatic decrease in thrombus formation was observed in animals that had been pre-challenged with the lower dose of tissue thromboplastin, whereas decrease in platelet count did not affect venous thrombosis under high thrombogenic challenge. When administered orally 2 hours before thrombosis induction, the ticlopidine analogue clopidogrel showed dose-dependent inhibition of thrombus formation in animals that were pre-challenged with a low dose of tissue thromboplastin (ED50 = 7.9 +/- 1.5 mg/kg, orally) but remained ineffective against high tissue thromboplastin-induced venous thrombosis. We further determined the effect of heparin and hirudin, and showed that both of these drugs exhibited a more potent antithrombotic activity after injection of the lower dose of tissue thromboplastin than after injection of a high dose of tissue thromboplastin. Therefore, using our model of stasis and hypercoagulability, platelet activation played a major role in the development of venous thrombosis when the thrombogenitic stimulus was mild.

Animals↗
PubMed1992

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.

Animals↗
PubMed1992

O-acylated heparin derivatives with low anticoagulant activity decrease proliferation and increase alpha-smooth muscle actin expression in cultured arterial smooth muscle cells.

Selectively O-acylated derivatives of various glycosaminoglycans were prepared and tested in vitro for their anticoagulant activity and their antiproliferative effect on rat and rabbit smooth muscle cells. When O-acylation (butyrylation or hexanoylation) had been performed on periodate-depolymerized heparin fragments having very low anticoagulant activity, the antiproliferative potency was markedly increased (IC50 = 2 and 1 micrograms/ml respectively, versus 31 micrograms/ml for starting compound) without an increase in anticoagulant activity. The antiproliferative activity was related to the degree of acylation. The O-acylated derivatives of heparin fragments were also very active in reversing the de-differentiation of smooth muscle cell in culture, as estimated by the increase in the expression of alpha-smooth muscle actin and alpha-smooth muscle actin mRNA.

Actins↗
PubMed1992

Importance of hepatic metabolism in the antiaggregating activity of the thienopyridine clopidogrel.

The thienopyridine clopidogrel is not active in vitro and must be administered i.v. or orally, suggesting that metabolism is necessary for activity. To verify whether the effect after i.v. administration was consecutive to recycling by hepatic bile secretion of clopidogrel or its metabolite(s) in the digestive tract, a catheter was implanted in the choledocus of rats, preventing bile and pancreatic secretions from being excreted into the digestive tractus. Two hours after clopidogrel administration (10 mg/kg, i.v.), blood was withdrawn and platelet-rich plasma aggregation was measured after the addition of 5 microM ADP. Clopidogrel treatment was equally efficient for sham-operated and catheterized animals (% inhibition of platelet aggregation: 76% and 59%, respectively) suggesting that the i.v. effect of clopidogrel was independent of re-absorption of biliary-excreted products and consequently that enteric metabolism is not necessary for activity. The antiaggregating activity of clopidogrel in rats before and after functional hepatectomy by a porto-jugular shunt was then studied. A great difference between treated animals was observed 30 min after i.v. administration of 25 mg/kg of clopidogrel. Per cent inhibition of platelet aggregation was 76% and 6% (P less than 0.001) for sham-operated and hepatectomized animals, respectively. Similar results were obtained after intraduodenal administration of clopidogrel, showing that the treatment was completely ineffective in hepatectomized animals. In isolated, blood-perfused rat livers, clopidogrel inhibited ADP-induced platelet aggregation, thereby supporting the theory that the activity of clopidogrel is highly dependent on hepatic metabolism.

Adenosine Diphosphate↗
PubMed1992

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.

Allergens↗
PubMed1992

Characterization of specific binding sites of 3H-labelled platelet-activating factor ([3H]PAF) and a new antagonist, [3H]SR 27417, on guinea-pig tracheal epithelial cells.

Binding of 3H-labelled platelet-activating factor ([3H]PAF) to guinea-pig tracheal epithelial cells was time-dependent, reversible and saturable. Scatchard analysis of the saturation-binding data indicated that [3H]PAF bound to one class of specific binding sites with high affinity (KD = 4.3 +/- 0.03 nM; Bmax. = 0.172 +/- 0.02 fmol/10(5) cells; n = 3). Unlabelled PAF competitively and selectively inhibited the specific binding of [3H]PAF with 50% inhibition at 4.8 +/- 0.07 nM (n = 3). SR 27417, the first member of a newly developed PAF antagonist series, competitively displaced [3H]PAF from its binding sites on guinea-pig tracheal epithelial cells with a Ki of 100 +/- 3 pM (n = 3). Studies carried out in parallel demonstrated that SR 27417 was 40 times more potent than C16-PAF itself and more than 100-fold as active as the best synthetic PAF-receptor antagonist yet described. [3H]SR 27417 displayed high-affinity, specific, reversible as well as saturable binding to a single class of binding sites on tracheal epithelial cells (KD = 94 +/- 7 pM; Bmax. = 0.181 +/- 0.04 fmol/10(5) cells; n = 3). C16-PAF, lyso-PAF, enantio-PAF, SR 27417 and other PAF-receptor antagonists had Ki values which were nearly identical for both [3H]PAF and [3H]SR 27417, demonstrating that in guinea-pig tracheal epithelial cells they have the same binding sites. In conclusion, these data suggest that tracheal epithelial cells contain PAF-specific receptors and indicate that SR 27417 is an extremely potent PAF-receptor antagonist, as well as being a suitable radioligand for labelling PAF receptors on intact cells.

Animals↗
PubMed1992

Induction of vascular smooth muscle cell growth by selective activation of the thrombin receptor. Effect of heparin.

The synthetic peptide, SFLLRNPNDKYEPF, has been recently described as a peptide mimicking the new amino-terminus created by cleavage of the thrombin receptor, therefore acting as an agonist of the thrombin receptor. This peptide was a potent mitogen for rabbit arterial smooth muscle cells (SMC) and exhibited the same activity as that of native alpha-thrombin. Both compounds stimulated the proliferation of growth-arrested SMCs with half-maximum mitogenic responses at 1 nM. NAPAP, a synthetic inhibitor of the enzymatic activity of thrombin, specifically inhibited thrombin-induced SMC growth (IC50 = 0.35 +/- 0.04 microM) but was without effect on the mitogenic effect of the agonist peptide. These results therefore demonstrate that the mitogenic effect of alpha-thrombin for SMCs is intimately linked to its esterolytic activity. Heparin, which inhibited fetal calf serum-induced SMC growth, was without effect on thrombin-induced SMC growth but strongly reduced the mitogenic effect of the agonist peptide (IC50 = 32 +/- 5 micrograms/ml). This effect was not related to the anti-coagulant activity of heparin but was highly dependent on molecular mass and on the global charge of the molecule and was also observed for other sulphated polysaccharides such as pentosan polysulphate.

Amino Acid Sequence↗
PubMed1992

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.

Administration, Oral↗
PubMed1992

Protective effect of SR 27417, a novel PAF antagonist, on PAF- or endotoxin-induced hypotension in the rat and the guinea-pig.

SR 27417, the first member of a newly-developed PAF antagonist series, inhibited in a dose-dependent manner the hypotensive effect of PAF in rats. It protected rats with an ED50 = 6 micrograms/kg, when given i.v., 1 min before PAF or p.o. (ED50 = 170 micrograms/kg) 1 h before PAF administration. After i.v. or oral administration, SR 27417 (1 and 3 mg/kg, respectively) exhibited extended duration of action (between 48 and 72 h). Similarly, i.v. administration of 1 to 6 mg/kg of SR 27417 afforded complete protection of guinea-pigs against endotoxin-induced hypotension but also totally reversed established endotoxin-induced hypotension. These results therefore confirm that PAF plays a major role in septic shock and that SR 27417 may be an effective prophylactic as well as a potent curative drug.

Administration, Oral↗
PubMed1992

Biochemical and pharmacological activities of SR 26831, a potent and selective elastase inhibitor.

SR 26831 ([[5-(2-chloro-benzyl-2-(terbutyloxycarbonyl)]-4,5,6,7- tetrahydrothieno(3,2-c)pyridine]N-oxide) is the first member of a new class of human leukocyte elastase inhibitors. SR 26831 inhibited in a dose-dependent manner elastases from human leukocytes or pancreas with IC50 values of 80 +/- 2.6 nM and 4.8 +/- 0.12 microM, respectively. Steady-state studies revealed that SR 26831 behaved like a noncompetitive, irreversible inhibitor of both types of enzymes. SR 26831 inhibited in a dose-dependent manner degradation of [3H]elastin and [3H]collagens (types I and IV) by human leukocyte elastase (IC50 values were between 1.2 and 1.8 microM). In this respect, SR 26831 was 3- to 20-fold more active than alpha-1-antitrypsin. SR 26831 was also highly selective for elastases inasmuch as it did not inhibit pepsin, collagenase, trypsin, alpha-chymotrypsin, factor Xa, plasmin, kallikrein, cathepsins B, C, D and G and thrombin. In the rabbit, SR 26831 was cleared rapidly from blood after i.v. injection, but affected intracellular leukocyte elastase activity shortly after either i.v. or p.o. administration. In the rat, i.v. or p.o. administration of SR 26831 prevented in a dose-dependent manner acute lung injury induced by intratracheal instillation of human leukocyte elastase. SR 26831 (1 mg/kg) was still efficient when it was administered 90 min before elastase instillation and was also able to limit further hemorrhage development in response to elastase, after it had begun. SR 26831 may therefore be of therapeutic value in the treatment of diseases such as rheumatoid arthritis or pulmonary emphysema thought to be due to the destructive action of leukocyte elastase.

Animals↗
PubMed1991

Protective effect of SR 27417, a novel PAF antagonist, on lethal anaphylactic and endotoxin-induced shock in mice.

In anaphylactic shock, SR 27417, the first member of a newly developed series of PAF (platelet-activating factor) antagonists, inhibited in a dose-dependent manner the lethal effect of antigen (ovalbumin) rechallenge in actively sensitized mice. It protected mice when given i.v. 5 min before ovalbumin challenge (ED50 = 50 micrograms/kg) or when given p.o. 1 h before ovalbumin administration (ED50 = 1.25 mg/kg). After i.v. or oral administration, SR 27417 (2.5 and 10 mg/kg, respectively) greatly improved the survival rate of mice after antigen challenge and had an extremely long duration of action (48 and 30 h, respectively). Similarly, i.v. or oral doses of SR 27417 afforded in mice complete protection against endotoxin-induced lethality (ED50 values were 100 and 150 micrograms/kg, respectively). SR 27417 (1 mg/kg) inhibited endotoxin-induced death in mice with impressive oral or i.v. durations of action of 66 and 110 h, respectively. These results confirm that PAF plays a major role in anaphylactic and endotoxin-induced shock and that SR 27417 may be an effective preventative drug.

Anaphylaxis↗
PubMed1991

Reversal of multidrug resistance by calcium channel blocker SR33557 without photoaffinity labeling of P-glycoprotein.

The altered pharmacology of drugs in multidrug-resistant cells (decreased accumulation and retention) appears to be mediated by a high molecular weight integral membrane protein, called P-glycogprotein (P-gp). Agents known to reverse this pleiotropic drug resistance (chemosensitizers) have been shown to interact with P-gp; and as such, the inhibition of photoaffinity labeling by P-gp probes (such as [3H]azidopine) has been proposed as a basis for mass screening of chemosensitizers. In this study, we provide direct evidence that a novel calcium channel blocker (SR33557), which was 4.5 times more potent in sensitizing P388/ADR cells to doxorubicin as compared to verapamil (while inducing a similar increase in uptake and decrease in efflux of [14C]doxorubicin, did not compete for the [3H]azidopine-binding site on P-gp, whereas verapamil did. Moreover, SR33557, which is inherently photoactivable, did not photolabel P-gp, but a 65-kDa protein did appear to be an acceptor; and this binding was displaced by diltiazem and nifedipine, but not by verapamil. Finally, the implication for the participation of a sphingomyelin/sphingosine cycle (as a potential lipid second messenger system) in the chemosensitization of P388/ADR cells was investigated. 30 microM SR33557 induced a 72% inhibition in acid lysosomal sphingomyelinase activity, a 5-fold increase in sphingosine levels, and a 75% inhibition in intracellular protein kinase C activity. Although no direct link is established between these observations and P-gp activity, further studies on a possible sphingosine-mediated regulation of P-gp may yield information on the involvement of this second messenger system in the action of SR33557.

Animals↗
PubMed1991

Tumor cell adherence to cultured capillary endothelial cells is promoted by activators of protein kinase C.

Stimulation of cells with protein kinase C (PKC)-specific activators such as phorbol esters increased in a reversible manner the rate of adherence of [3H]leucine-labelled L1210 cells to cultured bovine cerebral cortex capillary endothelial cells (CEC). This effect was not specific for L1210 cells since 12-O-tetradecanoyl phorbol 13-acetate (TPA) strongly increased the binding of various other tumor cell lines. Phorbol esters increased the rate of L1210 cell adhesion to CEC by enhancing their binding capacity without affecting the apparent affinity of L1210 cells for CEC. This stimulation was specific to the phorbol analogs which activate PKC since it was not effected by 4 alpha-phorbol didecanoate, known to be inactive for PKC. Down-regulation experiments showed that adhesion enhancement was entirely attributable to an effect on tumor cells without contribution of CEC intracellular PKC. PKC inhibitors like staurosporine, sphingosine and H-7 showed strong antagonistic activity towards TPA-induced L1210 cell adherence to CEC (IC50 = 0.5 nM, 160 nM and 10 microM, respectively). Adhesive proteins such as vitronectin, fibrinogen, fibronectin and the tetrapeptide RGDS, an active sequence from their cell-binding domains, exhibited potent, dose-dependent inhibition of PKC-induced tumor cell adhesion.

Animals↗
PubMed1991

Effect of pentosan polysulphate, standard heparin and related compounds on protein kinase C activity.

Ca2+/phospholipid-dependent protein kinase (PKC) was inhibited by sulphated polysaccharides. Pentosan polysulphate (PPS) and heparin were 8-10-times more potent than dextran sulphate or heparan sulphate. Steady-state studies revealed that PPS was a competitive inhibitor with respect to ATP with an apparent Ki value of 0.32 micrograms/ml and a non-competitive inhibitor with respect to histones. In contrast, the inhibition of PKC by heparin was competitive with substrate and non-competitive with respect to ATP. The interaction of sulphated polysaccharides with the catalytic domain of PKC was further demonstrated by the absence of effect on [3H]phorbol 12,13-dibutyrate binding to the regulatory domain of PKC. Furthermore, PPS and heparin inhibited equally cAMP-dependent protein kinase and tyrosine protein kinase. Structure-function relationships indicated that the Inhibition of protein kinases by PPS and heparin fractions was highly dependent on molecular weight. Additionally, PKC-affinity chromatography revealed that a high-molecular-weight heparin fraction with strong anti-PKC activity was eluted. We set out to demonstrate that heparin and PPS, which are potent antiproliferative agents on vascular smooth muscle cells (SMC), alter intracellular PKC activity (both membrane and cytosolic). Therefore, it is suggested that the mechanism by which sulphated polysaccharides inhibit SMC growth may be by direct inhibition of PKC in SMC.

Animals↗
PubMed1991

Characterization of extracellular binding sites for [3H]-staurosporine on capillary endothelial cells.

[3H]-staurosporine, a non-specific protein kinase inhibitor, bound with high affinity and in a reversible manner to specific and saturable binding sites in cultured bovine cerebral cortex capillary endothelial cells. Scatchard analysis revealed the presence of one class of non-interacting binding sites with an equilibrium dissociation constant (KD) of 9.2 nM and Bmax of 19.3 fmol/10(5) cells. The binding of [3H]-staurosporine was fully displaced by unlabelled staurosporine, H-7 and ATP with IC50 values of 6.9 nM, 3 microM and 0.4 microM respectively. Mild trypsinization of cells after [3H]-staurosporine binding revealed the presence of membrane-associated, extracellular binding sites which could be an ecto-protein kinase.

Alkaloids↗
PubMed1991

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)

Animals↗