Fred De Clerck

Inhibition of 5-hydroxytryptamine-induced and-amplified human platelet aggregation by ketanserin (R 41 468), a selective 5-HT2-receptor antagonist

Ketanserin, a selective 5-HT2-receptor antagonist, inhibits the reversible aggregation induced by 5-hydroxytryptamine (5-HT) in human platelet-rich plasma (PRP). In this respect, the compound is equipotent to cyproheptadine and more active than methysergide (IC50: 1.66×10−8M, 1.44×10−8M and 5.62×10−8M respectively). Ketanserin is active against 5-HT-induced platelet aggregation after bothin vitro and oral administration to human volunteers. At concentrations up to 500 times in excess of the IC50 for 5-HT-induced platelet reactions, ketanserin does not affect the aggregation induced by ADP, epinephrine, collagen or Thrombofax®, the prostaglandin biosynthesis of thrombin-stimulated platelets, nor the active uptake of14C-5-HT by platelets.5-Hydroxytryptamine amplifies the human platelet aggregation induced by threshold concentrations of ADP, collagen, epinephrine, norepinephrine and induced irreversible aggregation of platelets pre-sensitized with Thrombofax®. This amplification by 5-hydroxytryptamine results in a platelet response typical for the potentiated agonist; for the combination of the monoamine with collagen, the serotonergic amplification results in enhanced aggregation, release of β-TG and PF4 and excessive formation of TXB2. Ketanserin, after bothin vitro and oral administration to man reduces the amplified response to the level of the potentiated agonist.The present evidence suggests the presence of functional 5-HT2 receptors on the human platelet, different from those involved in the uptake of the monoamine.

Maternal food restriction in the second half of pregnancy affects vascular function but not blood pressure of rat female offspring

Food restriction during pregnancy in rats induces intrauterine growth retardation with consequences persisting into adulthood. In the present study we have investigated the hypothesis that malnutrition in pregnant rats may lead to altered cardiovascular function in adult female offspring. Perinatal growth retardation was induced by a 50% reduction of normal dietary intake in rats during the second half of pregnancy. Systolic and diastolic blood pressure values and heart rate were recorded in conscious female offspring (100 d old) using a femoral artery probe. No significant differences in heart rate, or in systolic and diastolic blood pressures were recorded between control offspring and offspring of nutritionally deprived rats. In order to ascertain whether cardiovascular variables in the offspring were influenced by lactation, subgroups of offspring from food-restricted dams were fostered with lactating dams fed on a normal diet. Blood pressure and heart rate were also found to be normal in these offspring. The rise in blood pressure associated with NO inhibition was similar in all groups. Isolated resistance artery function was assessed in vitro in offspring (100-120 d old) of a second group of semi-starved dams. Small mesenteric arteries from these animals showed reduced endothelium-dependent relaxation (to acetylcholine and bradykinin), but enhanced sensitivity to exogenous NO (sodium nitroprusside). We conclude that food restriction during the second half of pregnancy and/or lactation does not induce hypertension in adult offspring, but may effect subtle changes in vascular function.

Demonstration of S2-receptor binding sites on cat blood platelets using [3H]ketanserin

[3H]Ketanserin showed substantial binding to cat platelet membranes; 40% of this was specific binding, characterized by a plateau in serotonin antagonist inhibition curves, and another 40% was non-specific binding to structural recognition sites displaceable by unlabelled ketanserin. Specific [3H]ketanserin binding to platelets shows the features of serotonin S2-receptor binding sites previously identified in rat pre-frontal cortex and in striatum. In platelets, KD = 1.02 nM and Bmax = 86 fmol/10(9) platelets; the Ki values of 21 compounds were similar in the three different tissues.

Overexpression of endothelial nitric oxide synthase suppresses features of allergic asthma in mice

BackgroundAsthma is associated with airway hyperresponsiveness and enhanced T-cell number/activity on one hand and increased levels of exhaled nitric oxide (NO) with expression of inducible NO synthase (iNOS) on the other hand. These findings are in paradox, as NO also relaxes airway smooth muscle and has immunosuppressive properties. The exact role of the endothelial NOS (eNOS) isoform in asthma is still unknown. We hypothezised that a delicate regulation in the production of NO and its bioactive forms by eNOS might be the key to the pathogenesis of asthma.MethodsThe contribution of eNOS on the development of asthmatic features was examined. We used transgenic mice that overexpress eNOS and measured characteristic features of allergic asthma after sensitisation and challenge of these mice with the allergen ovalbumin.ResultseNOS overexpression resulted in both increased eNOS activity and NO production in the lungs. Isolated thoracic lymph nodes cells from eNOS overexpressing mice that have been sensitized and challenged with ovalbumin produced significantly less of the cytokines IFN-γ, IL-5 and IL-10. No difference in serum IgE levels could be found. Further, there was a 50% reduction in the number of lymphocytes and eosinophils in the lung lavage fluid of these animals. Finally, airway hyperresponsiveness to methacholine was abolished in eNOS overexpressing mice.ConclusionThese findings demonstrate that eNOS overexpression attenuates both airway inflammation and airway hyperresponsiveness in a model of allergic asthma. We suggest that a delicate balance in the production of bioactive forms of NO derived from eNOS might be essential in the pathophysiology of asthma.

Blood platelets in human essential hypertension

Blood platelets of patients with essential hypertension display signs of both increased sensitivityin vitro to aggregating stimuli believed to contribute to thrombosis and of activationin vivo possibly expressing the release of vasoactive products. The mean features of the modified platelet profile in hypertension include an increased α2-adrenergic receptor density, an enhanced rate of adhesion/aggregation in particular in response to ADP and arachidonic acid, a greater sensitivity for thrombin and adrenaline to stimulate increases in cytoplasmatic-free Ca2+, increased resting levels of cytoplasmatic-free Ca2+, a reduced content of serotonin often combined with a defective uptake mechanism, a facilitated efflux rate of noradrenaline, an exaggerated release reactionin vivo as indicated by the increased plasma levels of Betathromboglobulin and a shortened platelet life span. These changes occur to various extents in some, but not all, hypertensive patients and are not always strictly related to the degree of blood pressure increase. On the contrary, platelet cyclooxygenase and thromboxane synthetase activity are in the normal range.

Platelet-mediated vascular contractions: Inhibition of the serotonergic component by ketanserin

Abstract In vitro stimulation of washed rat platelets (2.5 x 1010/1) with thrombin (0.002 U/l) as well as their activation on de-endothelialized vascular preparations, produced a strong contraction of isolated rat caudal arteries. Pharmacodissection and specific measurements of various mediators (5-hydroxytryptamine, thromboxane B2, ATP) showed the platelet-mediated contraction to be mainly mediated by 5-hydroxytryptamine, either by its direct effect or through amplification of prostaglandin-effects at the level of the vessel or the platelets. Platelet-mediated vasospastic episodes were effectively reduced by ketanserin, a selective 5-HT2 receptor antagonist.

Does the antiarrhythmic effect of ischemic preconditioning in rats involve the L-arginine nitric oxide pathway ?

Ischemic preconditioning (PC) has been shown to limit ischemia- and reperfusion-induced arrhythmias. We wished to determine whether the antiarrhythmic effect of PC would be affected by inhibition of the L-arginine nitric oxide (NO) pathway in anesthetized rats. Ischemia and reperfusion were produced by occlusion and release of a snare around the left coronary artery in all rats. The effect of PC (three cycles of 2-min coronary artery occlusion and 5-min reperfusion) on development of reperfusion-induced arrhythmias after 5-min coronary artery occlusion was studied in 12 rats. In 24 other rats, the specific NO synthesis inhibitor NG-monomethyl-L-arginine (L-NMMA 10 mg/kg, n = 12) or the muscarinic receptor antagonist-NO synthesis inhibitor nitro-L-arginine methyl ester (L-NAME 10 mg/kg, n = 12), was administered intravenously (i.v.) before PC. In control groups, solvent (n = 15), L-NAME (10 mg/kg i.v., n = 12), L-NMMA (10 mg/kg i.v., n = 12), or L-arginine (L-Arg 100 mg/kg i.v., n = 12) was administered to rats 5 min before coronary artery occlusion without PC. PC significantly reduced the incidence of ventricular premature beats (VPBs) from 100% in the non-PC solvent group to 17%, decreased the incidence of ventricular tachycardia (VT) from 93 to 8%, and abolished the incidence of reversible and irreversible ventricular fibrillation (RVF and IVF: 87 and 47% in the non-PC solvent group, respectively). L-NAME and L-NMMA did not significantly affect the protective effect of PC on reperfusion-induced arrhythmias.(ABSTRACT TRUNCATED AT 250 WORDS)

Ischemia/reperfusion-induced arrhythmias in anaesthetized rats : a role of Na+ and Ca2+ influx

We hypothesized that by limiting the Na+ and Ca2+ loading by a blocker/inhibitor of the Na+ channel (lidocaine), Na+ overload (R56865: N-[1-[4-(4-fluorophenoxy)butyl]-4-piperidinyl]-N-methyl-2-benzothiazo lamine), Ca2+ channel (verapamil), Na+ -H+ exchange (ethylisobutyl amiloride) or of Na+ -Ca2+ exchange (No. 7943: 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate), it should be possible to reduce ischemia/reperfusion-induced arrhythmias. To test this hypothesis, we used anaesthetized rats subjected to 5 min of coronary artery occlusion followed by 10 min of reperfusion to study antiarrhythmic effects of above compounds on reperfusion-induced ventricular premature beats, ventricular tachycardia, and reversible and irreversible ventricular fibrillation. Compound or saline was administered as an intravenous bolus injection at 5 min before ischemia. Pretreatment with lidocaine (5 mg/kg), verapamil (0.63 mg/kg), R56865 (0.63 mg/kg) or ethylisobutyl amiloride (1.25 mg/kg) significantly reduced or abolished all types of ventricular arrhythmias. However, pretreatment with verapamil was associated with second or third degree heart block in 3 out of 12 animals. Pretreatment with No. 7943 did not significantly influence the ischemia/reperfusion-induced ventricular arrhythmias. The present results suggest that both intracellular Na+ -and Ca2+ -loading play important roles in reperfusion-induced ventricular arrhythmias and the inhibition of Na+ -Ca2+ exchange to limit Ca2+ loading probably does not play any important role in ischemia/reperfusion-induced arrhythmias in anaesthetized rats.

Direct evidence for the contractile capacity of endothelial cells.

Abstract Dog aorta-derived endothelial cells, grown in culture and resuspended in normal human or canine citrated platelet-free plasma, invariably retracted the fibrin clot when stimulated with exogenously added or endogenously formed thrombin. The onset, rate and extent of this retraction depended upon the cell density, the concentration of thrombin, the cellular integrity, the homogeneous cell distribution and the availability of free extracellular Ca 2+ . Endothelial cell-induced clot retraction was completely inhibited by EGTA, by VK 774-induced phosphodiesterase inhibition and by combined treatment with PGE 1 and papaverine. It was partly reduced by nocodazole-induced microtubule disruption, by cytochalasin B, by verapamil- and flunarizine-induced blockade of Ca 2+ -fluxes but was not affected by cyclo-oxygenase inhibitor suprofen. Incorporation of additional Ca 2+ into non-retracting, Reptilase®-formed clots resulted in a retraction which was not blocked by heparin. In addition of 5-hydroxytryptamine, ADP, histamine or epinephrine slightly enhanced this retraction.

The involvement of 5-HT2-receptor sites in the activation of cat platelets.

5-Hydroxytryptamine (5-HT) induces a concentration-dependent aggregation/release of/by cat platelets (Km = 6.2 x 10(-7) M); this activation is inhibited (Ki = 5.24 x 10(-9) M) or reversed by ketanserin, a selective 5-HT2 receptor antagonist. Comparison of the inhibition of specific [3H] ketanserin binding to cat platelet membranes and rat pre-frontal cortex membranes with that of 5-HT-induced aggregation of cat platelets obtained with various drugs, displaying various receptor binding profiles, reveals a highly significant correlation between the ligand binding and the physiological response (Spearman correlation coefficient r = 0.92 and r = 0.91 respectively, p less than 0.0001; n = 14); inhibition of platelet activation by 5-HT and of uptake of 5-HT are not correlated. Secondary aggregation induced by ADP as well as collagen-induced aggregation are inhibited by the 5-HT receptor antagonists suggesting a primary role of 5-HT in the secondary platelet recruitment subsequent to a release reaction. This study demonstrates a functional role for the 5-HT2 receptors in the primary activation of the platelets by 5-HT and in the secondary aggregation induced by other agonists, especially in platelets superreactive to 5-HT2 receptor activation.