Involvement of inositol 1,4,5-triphosphate and protein kinase C in thrombin-induced contraction of porcine pulmonary artery.

The role of the intracellular messengers inositol 1,4,5-triphosphate (IP3) and protein kinase C (PKC) in the thrombin (3 U/mL)-induced contraction of endothelium-denuded porcine pulmonary arteries was investigated. Thrombin induced a sustained contractile response with an initial transient increase in IP3 to about 160% of the unstimulated control. Omission of extracellular Ca2+ or preincubation with verapamil (10 mumol/L) reduced the maximum of contraction without significantly affecting the thrombin-induced increase in IP3. To evaluate the role of PKC for the contractile response, the PKC was activated directly by phorbol 12,13-dibutyrate (PDBu, 50 nmol/L). The phorbol ester produced a slowly increasing tonic contraction without any changes in the basal IP3 level. There was a moderate inhibition of PDBu-induced contractions in Ca(2+)-free solution, while they were not inhibited after preincubation with verapamil. Preincubation with the PKC inhibitor staurosporine (50 nmol/L) significantly reduced the PDBu-induced contraction (by about 80%). In thrombin-stimulated vessels staurosporine only inhibited the tonic phase of the contractile response whereas the increase in IP3 and the phasic component of contraction were still evident. These results suggest that IP3 and PKC are involved in the thrombin-induced contraction. The phasic component of contraction is associated with the generation of IP3; the tonic component might be due to the activation of PKC.

Defibrinogenation with benzoyl-batroxobin.

The B. moojeni thrombic protease, batroxobin, was acylated by 4-amidinophenyl benzoate at the active site serine hydroxyl. From the enzymatically inactive benzoyl-batroxobin, batroxobin is generated with a half-life of deacylation of about one hour. The clotting activity of benzoyl-batroxobin in plasma is recovered with deacylation. The effects of batroxobin and benzoyl-batroxobin were studied following intravenous injection in rats. Compared to defibrinogenation with batroxobin, that obtained with benzoyl-batroxobin was much retarded. Batroxobin caused microthrombosis initially, which did not develop upon injection of benzoyl-batroxobin in equivalent doses.

A synthetic inhibitor of factor Xa, DX-9065a, reduces proliferation of vascular smooth muscle cells in vivo in rats.

The effect of factor Xa inactivation on the proliferation of vascular smooth muscle cells in vivo was investigated in an experimental restenosis model in rats by using the direct factor Xa inhibitor DX-9065a. In the left common carotid artery, an injury of the vascular endothelium was produced by four external vessel clamps for 60 minutes. After 14 days, 3H-labeled methyl thymidine and 5-bromo-2'-deoxyuridine, respectively, were injected intraperitoneally. After 24 hours, both the left (damaged) and right (nondamaged) carotid arteries were removed, and the incorporation of 3H-methyl thymidine/microg protein was determined. For morphological analysis, the cells were labeled with hematoxylin as well as 5-bromo-2'-deoxyuridine. Stained vascular smooth muscle cell nuclei were counted, and the proliferation index (percentage of 5-bromo-2'-deoxyuridine-positive nuclei to total nuclei stained with hematoxylin) was determined. An external damage of the carotid artery induced proliferation of vascular smooth muscle cells and formation of a neointima within 2 weeks after vessel injury. As compared with control animals, single subcutaneous injection of DX-9065a (2.5, 5, and 10 mg/kg) given 30 minutes before vessel injury significantly reduced the incorporation of 3H-methyl thymidine/microg protein and the total cell number, as well as the proliferation index. The antiproliferative action of DX-9065a was not dose dependent in the range from 2.5 to 10 mg/kg s.c. A combination of bolus injection (5 mg/kg s.c.) with continuous administration (5 mg/kg/d s.c. for 7 and 14 days, respectively) did not increase the antiproliferative effect of DX-9065a. The results indicate a role of factor Xa in the complex pathogenesis of restenosis and the usefulness of a highly effective and selective inhibitor of factor Xa to inhibit proliferative processes.

Relaxant and contractile responses of porcine pulmonary arteries to a thrombin receptor activating peptide (TRAP).

Recent studies on cloning of the thrombin receptor, which belongs to the family of G-protein-coupled receptors, suggest that thrombin cleaves a peptide from the extracellular N-terminus. A synthetic peptide of 14 amino acids corresponding to the sequence of the newly generated N-terminus was found to possess thrombin-like activity in several cells endowed with thrombin receptors. The relaxant and contractile effects of this thrombin receptor activating peptide (TRAP, Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe) were investigated in porcine pulmonary arteries and compared with the action of thrombin. In PGF2 alpha-precontracted vessels with intact endothelium, TRAP (0.3-10 mumol/l) caused reversible transient and concentration-dependent relaxation which was absent after mechanical removal of the endothelium. Preincubation of the vessels with NG-nitro-L-arginine (200 mumol/l) markedly reduced the relaxation. The TRAP-induced relaxation was associated with an increase in cGMP in the arteries. In comparison to thrombin, TRAP (EC50: 0.8 mumol/l) was less potent by more than three orders of magnitude. In endothelium-denuded pulmonary arteries TRAP (1-20 mumol/l) caused a concentration-dependent contraction which was reversible after washout. The TRAP-induced contractile response was preceded by an increase in generation of inositol 1,4,5-triphosphate (IP3); the peak of IP3 accumulation was reached after 30 s. Compared with the contractile effect of thrombin, that of TRAP was weaker by three of magnitude. The vascular effect of TRAP was not inhibited by the thrombin inhibitors hirudin or heparin while the protein kinase C inhibitor staurosporine (0.1 mumol/l) preferentially inhibited the tonic phase of contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Contractile effects of thrombin in porcine pulmonary arteries and the influence of thrombin inhibitors.

Thrombin catalyzes not only the conversion of fibrinogen to fibrin but also activates several receptor-mediated cell responses. In ring segments of porcine pulmonary arteries the contractile effect of thrombin was studied in the presence and absence of endothelium. The integrity of endothelium was assessed by the bradykinin-induced relaxation of PGF2 alpha (3 mumol/l)-precontracted vessels which was absent after mechanical removal of endothelium. Thrombin at 0.1 to 10 U/ml (i.e. about 1-100 nmol/l) caused a sustained contraction in endothelium-denuded arteries with a maximum at 20-30 min. In vessels with intact endothelium a significant increase in tension up to 1 U/ml was observed preceded by a transient relaxant response. The contractile effect in vessels with intact endothelium was comparatively weaker. This is probably due to the release of EDRF from endothelial cells since blockade of EDRF synthesis by NG-nitro-L-arginine augmented the thrombin-induced contractions in arteries with intact endothelium. Indomethacin did not alter the contractile effect. However, in vessels with endothelium and in endothelium-denuded vessels the contractions were reduced when extracellular calcium was omitted. Verapamil (10 mumol/l) significantly diminished the contractile effect only in endothelium-denuded vessels. On preincubation of endothelium-denuded arterial ring segments with myo-[2-3H]inositol the addition of thrombin (10 U/ml) caused an accumulation of [3H]inositol-1,4,5-triphosphate (IP3). A maximum was observed after 2 min preceding the maximum increase in contraction. Measurement of thrombin-induced endogenous IP3 generation by radioreceptor assay yielded the same results. The thrombin-induced contractile effect requires the proteolytic activity of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of thrombin-induced contractile responses by protein kinase inhibitors in porcine pulmonary arteries.

The clotting enzyme thrombin is known to cause receptor-mediated contractile effects in isolated blood vessels. In the present studies the influence of protein kinase inhibitors on the contractile response of porcine pulmonary arteries to thrombin (3 U/ml) was investigated. Endothelium-denuded rings (2-3 mm) from small arteries were placed in organ baths for isometric tension recording. The vessels were preincubated for 30 min with the inhibitors before inducing contractions. In the presence of the protein kinase C (PKC)-inhibitors staurosporine, BIM I (bisindolyl-maleimide I), chelerythrine and Ro 31-8220 (1 microM each), the contractile responses to the PKC activator phorbol 12,13-dibutyrate (PDBu; 50 nM) were diminished by 70-100%. However, for inhibition of thrombin-induced contractions generally higher concentrations of the inhibitors were required. Only staurosporine at 1 microM inhibited the thrombin effect by about 75%. The tyrosine kinase inhibitor erbstatin (30 microM) did not significantly alter the thrombin effect, whereas genistein at 10 microM caused a significant inhibition of contractile responses to both thrombin and PGF2alpha. At 100 microM genistein also inhibited the contractile effects of PdBu and KCl. These studies suggest that activation of both PKC and non-receptor tyrosine kinases seems to be involved in the signal transduction pathways of thrombin-induced contractile effects in isolated vessels.

On the local anaesthetic action of propolis and some of its constituents.

An ethanolic propolis extract and some constituents isolated from propolis were tested on the cornea of the rabbit and of the mouse for local anaesthetic activity. Total anaesthesia was obtained with the total extract as well as with the compounds 5,7-dihydroxyflavanone (pinocembrin), 5-hydroxy-7-methoxyflavanone (pinostrobin) and with a mixture of caffeic acid esters. Each of these compounds was nearly thrice as potent as the total extract. Propoxypiperocaine which was tested for the purpose of comparison was still efficient in an almost 10-fold lower concentration. When applied subcutaneously, pinocembrin and the mixture of caffeic acid esters produced nearly the same anaesthesia as lidocaine.

Pharmacological characterization of a new structural variant of 4-amidinophenylalanine amide-type synthetic thrombin inhibitor.

The synthetic thrombin inhibitor N alpha-(2-naphthylsulfonylglycyl)-4-amidinophenylalanyl-proline (1) was synthesized in order to evaluate the importance of the carboxyl group in its amine portion for fundamental pharmacodynamic and pharmacokinetic properties of these benzamidine derivatives. The compound is better tolerated in mice and rats than are similar compounds lacking this carboxyl group. It has a significantly longer plasma half-life in rabbits compared to the corresponding compound bearing piperidine instead of proline in the amide moiety. The main excretory route of 1 is via the bile. Hepatic uptake and/or biliary excretion show, however, another time course than that seen for the piperidide. Lower toxicity and more suitable pharmacokinetics may compensate for the loss of thrombin inhibitory potency of this new synthetic thrombin inhibitor.

[On the antimicrobial activity of propolis and propolis constituents (author's transl)]. / Zur antimikrobiellen Wirksamkeit von Propolis und Propolisinhaltsstoffen.

After a survey of the literature on the antimicrobial activity of the bee product propolis, the authors discuss their own findings as compared to the chemotherapeutical agents streptomycin, oxytetracycline, chloramphenicol, nystatin, griseofulvin and sulphamerazine. According to the results obtained by testing 25 isolated constituents on Bacillus subtilis, Staphylococcus aureus, Candida albicans and Trichophyton mentagrophytes, the antimicrobial properties of this mixture of natural substances are mainly attributable to the flavonoids pinocembrin, galangin, pinobanksin, pinobanksin-3-acetate as well as to the p-coumaric acid benzyl ester and a caffeic acid ester mixture. None of the isolated substances was as potent as the antibiotics tested for the purpose of comparison. The relatively good antimycetic activity of the 5,7-dihydroxyflavanone pinocembrin seems noteworthy. Finally, possible mechanisms of the antimicrobial action of the flavonoids are discussed.

[On the use of the relative surface tension xi in studies on the structure-activity relationships (author's transl)]. / Zur Anwendung der relativen Oberflächenspannung xi in der Struktur-Wirkungs-Analyse.

The relative surface tension xi = (Pi/Mv)4 can be used in structure-activity studies as additional parameter. Here, Pi = parachor, Mv = molar volume (Tables 1 and 2) of the substituents. Some examples demonstrate the application.

Isolation and characterization of a thrombin inhibitor from the tick ixodes ricinus.

The secretion of the salivary glands of ticks, Ixodes ricinus, contains anticoagulant substances. Some years ago an antithromboplastin, ixodin, was described. The present paper refers the isolation and characterization of a second anticoagulant substance of the ticks named ixin. Ixin proved to be a relatively stable and specific thrombin inhibitor. The multi step procedure for isolation results in a 850-fold purification. The preparation obtained has a specific activity of 250 antithrombin units/mg. It is not homogeneous and still contaminated. The substance was assumed to be a miniprotein.

Defibrinogenation by batroxobin and acylated batroxobin in rats.

The thrombin-like snake venom enzyme, batroxobin, was acylated by 4-amidinophenyl benzoate at the active site serine hydroxyl. From the enzymatically inactive benzoyl-batroxobin, batroxobin is generated with a half-life of deacylation of about 1 hour. The clotting activity of benzoyl-batroxobin in plasma is recovered with deacylation. The effect of batroxobin and benzoyl-batroxobin were studied following intravenous injection in rats. Compared to defibrinogenation with batroxobin, that obtained with benzoyl-batroxobin was much retarded. Batroxobin caused microthrombosis initially, which did not develop upon injection of benzoyl-batroxobin in equivalent doses.

Inositol 1,4,5-triphosphate and protein kinase C are involved in thrombin- and trap-induced vascular smooth muscle contraction.

Thrombin (30 nmol/l) as well as the thrombin receptor activating peptide (TRAP), 10 mumol/l) induce a sustained contraction of endothelium-denuded porcine pulmonary arteries. The first phasic component of contraction is associated with the generation of IP3 which precedes the development of contractile force. Since the PKC inhibitor staurosporine (50 nmol/l) completely inhibits the tonic contraction this component of contraction seems to be due to the activation of protein kinase C (PKC). The thrombin- and TRAP-induced vasoconstriction strongly depends on extracellular calcium; the remaining thrombin- or TRAP-induced contraction in Ca(2+)-free medium seems to be attributed to the IP3-mediated release of calcium from intracellular stores.

Relaxant and contractile responses of porcine pulmonary arteries to thrombin and thrombin receptor activating peptides.

The vascular effects of thrombin and thrombin receptor activating peptides (TRAP) were studied on isolated rings from porcine pulmonary arteries. In prostaglandin F2 alpha (PGF2 alpha)-precontracted vessels with intact endothelium, both thrombin- and TRAP-induced nitric oxide-mediated relaxation, whereas in endothelium-denuded vessels thrombin and TRAP elicited concentration-dependent contractile responses. The first phasic component of contraction was associated with increased generation of inositol 1,4,5-triphosphate and the tonic component seemed to be due to the activation of protein kinase C. Both peptides (TRAP-6 with 6 and TRAP-14 with 14 amino acid residues) did not differ in their intrinsic activity; like thrombin, both peptides elicited dualistic vascular effects but their potency was more than three orders of magnitude less than that of thrombin.

Pharmacological activities of a homologous series of pyrazole derivatives including quantitative structure-activity relationships (QSAR).

In a series of fourteen 1-benzoyl-3-methyl-pyrazole derivatives the antiinflammatory activity in the carrageenin edema of the rat paw was found to be in the potency range of that of aminophenazone. This holds also true of the analgesic, anticonvulsive, and anticholinergic activity of the compounds whereas antihistaminergic activity was 10-fold better and acute toxicity was less than that of aminophenazone. Using the MASCA model as a multivariate method in QSAR the regression matrix of the derivatives substituted in p-position of the benzoyl moiety has been calculated. According to that, all of the biological activities are mainly described in terms of pi, sigma, xi, and L. The increase of inflammatory activity could be expected by substitution of electro-positive substituents of short length (L) and little relative surface tension (xi). Concomitantly, analgesic and anticonvulsive activities could increase whereas anticholinergic and antihistaminergic activities are expected to decrease. The HANSCH analysis is surely to be preferred in investigations of the relationship between physicochemical parameters of compounds and a certain biological activity evoked by them. However, the multivariate MASCA model could be of some advantage in the optimization of chemical structure against the whole profile of biological activities in a series of compounds. Some problems of the MASCA model are briefly discussed.

Thrombin receptor activating peptide-induced cellular effects: comparative studies on human platelet activation and endothelium-dependent relaxation of porcine pulmonary arteries.

The thrombin receptor activating peptides with 6 and 14 amino acids (TRAP-6,TRAP-14) caused aggregation of washed platelets as well as of platelets in citrated and hirudin plasma. Stimulation of platelets was associated with an increase in cytosolic Ca2+ and formation of thromboxane. In porcine pulmonary arteries they induced reversible endothelium-dependent relaxation of precontracted vessels via release of endothelium-derived nitric oxide. TRAP-6 and TRAP-14 did not differ in their intrinsic activity. Both peptides possess thrombin-like activity, but their potency is more than three orders of magnitude lower than that of thrombin.

Biliary excretion of synthetic benzamidine-type thrombin inhibitors in rabbits and rats.

In rabbits and rats the excretion via the bile of the highly effective and selective synthetic thrombin inhibitor N alpha-(2-naphthylsulfonylglycyl)-4-amidinophenylalanine piperidide was studied after various routes of administration and varying doses. The concentration in the bile was determined using a biological method, the reliability of which was checked by HPLC-determinations. The compound was excreted in the bile in biologically active unchanged form. Cumulative biliary excretion of the synthetic thrombin inhibitor in rabbits amounted to a high percentage of the administered dose after low doses given either systemically or intraportally. In rats the excretion rates during and after intravenous infusion reached a maximum indicating saturation kinetics. Hepatic uptake and biliary excretion of the highly basic benzamidine derivative essentially contribute to its short plasma half-life.