The effects of a novel sulphidopeptide leukotriene antagonist, BAY x7195, against elicited bronchoconstriction in the anaesthetized guinea-pig.

1. The novel leukotriene antagonist Bay x7195, has been evaluated against bronchoconstriction induced by leukotriene D4 (LTD4), the thromboxane A2 (TXA2) mimetic U46619, histamine and antigen, in the guinea-pig in vivo by use of a modified Konzett-Rössler preparation. 2. LTD4, given intravenously (i.v.) at 1 or 3 microg kg(-1) in the presence of indomethacin and sotalol, caused a 50-70% maximal bronchoconstriction in most animals. 3. BAY x7195, given i.v., orally (p.o.), by aerosol or dry powder insufflation, in lactose, reduced LTD4-induced bronchoconstriction dose-dependently. The approximate ID50 values were 83 microg kg(-1), 3 mg kg(-1), 0.0003% w/v for 20 breaths and 20 microg respectively. 4. The action of BAY x7195 (10 mg kg(-1), p.o.) was long lasting, causing significant inhibition of the LTD4-induced response (88% reduction) 8 h after dosing. 5. When given intravenously, in the presence of selected antagonists, BAY x7195 caused a dose-related reduction in the antigen-induced response, with an approximate ID50 of 2 mg kg(-1). 6. At 3 mg kg(-1), i.v., a dose which abolished the response to LTD4, BAY x7195 had no effect on U46619- or histamine-induced bronchoconstriction. 7. BAY x7195 is a potent, selective and long acting antagonist of LTD4-induced bronchoconstriction, in an anaesthetized, ventilated guinea-pig model. It is therefore worthy of clinical evaluation in diseases believed to involve the sulphidopeptide leukotrienes, such as asthma.

Leukotriene receptors on human pulmonary vascular endothelium.

1. Cysteinyl-leukotrienes cause contractions and/or relaxations of human isolated pulmonary vascular preparations. Although, the localization and nature of the receptors through which these effects are mediated have not been fully characterized, some effects are indirect and not mediated via the well-described LT1 receptor. 2. In human pulmonary veins (HPV) with an intact endothelium, leukotriene D4 (LTD4) induced contraction above basal tone. This response was observed at lower concentrations of LTD4 in the presence of nitric oxide synthase inhibitor N omega-nitro-L-arginine (L-NOARG). Contractions (in the absence and presence of L-NOARG) were partially blocked by the LT1 antagonists (MK 571 and ICI 198615). 3. LTD4 relaxed HPV previously contracted with noradrenaline. This relaxation was potentiated by LT1 antagonists, but was abolished by removal of the endothelium. LTD4 also relaxed human pulmonary arteries (HPA) precontracted with noradrenaline but this effect was not modified by LT1 antagonists. 4. The results suggest that contraction of endothelium-intact HPV by LTD4 is partially mediated via LT1 receptors. Further, in endothelium-intact HPV, this contraction was opposed by a relaxation induced by LTD4, dependent on the release of nitric oxide, which was mediated, at least in part, via a non-LT1 receptor. In addition, LTD4 relaxation on contracted HPA was not mediated by LT1 receptors. 5. The mechanical effects of LTD4 on human pulmonary vasculature are complex and involve both direct and indirect mechanisms mediated via at least two types of cysteinyl-leukotriene receptors.

Characterisation of leukotriene receptors on rat lung strip.

The leukotriene receptor(s) present on rat lung strip have been characterised using the natural agonists, a selective mimetic, and potent (cysLT1) selective leukotriene receptor antagonists. Leukotriene C4 and leukotriene D4 displayed comparable contractile potencies whilst leukotriene E4 was less potent. However, both leukotriene D4 and leukotriene E4 were found to be partial agonists relative to leukotriene C4. Responses to all three leukotrienes were competitively antagonised by ICI 198615 (1-((2-methoxy-(4-phenylsulfonyl)-aminocarbonyl)-phenyl) methyl)-1H-indazol-6-yl) carbonic acid cyclopentyl ester), SK&F 104353 (2-(R)-hydroxy-3(S)-(2-carboxyethylthio)-3-(2-[8-phenyloctyl ]- phenyl)propanoic acid, and MK571 (+/-(E)-3-[3-[2-(7-chloro-2-quinolin-yl)ethenyl]-phenyl)- ([3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]thio]propanoic acid) with comparable affinities irrespective of the agonist used. This indicates that rat lung contains a homogeneous population of leukotriene receptors and that they are of the CysLT1 type.

BAY u9773, a novel antagonist of cysteinyl-leukotrienes with activity against two receptor subtypes.

The effects of BAY u9773 (6(R)-(4'-carboxyphenylthio)-5(S)-hydroxy-7(E),9(E), 11(Z),14(Z)-eicosatetraenoic acid), a cysteinyl-leukotriene analogue, were investigated on a variety of smooth muscle preparations in order to determine its profile as a cysteinyl-leukotriene receptor antagonist. The tissues were contracted with leukotriene C4 or leukotriene D4 and their receptor characteristics defined as either 'typical' or 'atypical' according to the activity or inactivity, respectively, of the selective antagonists ICI 198615, MK 571 and SKF 104353. BAY u9773 antagonised 'typical' cysteinyl-leukotriene receptors with pA2 (or pKB) values in the range 6.8-7.4 and also antagonised 'atypical' receptors with pA2 values in the range 6.8-7.7. However, BAY u9773 had no effect at 10(-6) M against a selection of non-leukotriene stimuli in the same preparations. BAY u9773 competitively displaced [3H]leukotriene D4 binding to guinea-pig lung homogenate, with a pKi of 7.0 +/- 0.1. In the guinea-pig lung strip, BAY u9773 was found to be inactive at 10(-6)M against leukotriene C4- and leukotriene D4-induced contractions, which may suggest the existence of a third type of cysteinyl-leukotriene receptor. These data demonstrate that BAY u9773 is a selective cysteinyl-leukotriene receptor antagonist with comparable activity at both 'typical' and 'atypical' receptors and as such represents a valuable tool for the study of cysteinyl-leukotriene receptors.

Characterisation of the peptido-leukotriene receptor PL2 on the ferret spleen strip.

The peptido-leukotriene receptor(s) (PL) on the ferret isolated spleen strip have been characterised by functional studies using the naturally occurring leukotrienes (LTs), a range of structurally distinct PL antagonists, and by ligand binding studies. LTB4 (0.01-10 microM) was inactive on ferret spleen whereas LTC4, LTD4 and LTE4 produced concentration-related contractions with maximal responses, relative to noradrenaline, of 57% (EC50 0.28 microM), 60% (EC50 0.5 microM) and 7% respectively. The leukotriene responses were unaltered by L-serine borate, L-cysteine, indomethacin, phentolamine, propranolol, mepyramine, methysergide or atropine, suggesting that the peptido-leukotrienes were acting through distinct PL receptors. The PL1 antagonists, FPL 55712 (0.01-10 microM), ICI 198615 (10 microM), SK&F 104353 (10 microM) and MK541 (10 microM) were all inactive against LTC4- or LTD4-induced contractile responses. LTE4 was a partial agonist with respect to LTC4 and LTD4 with pKB values of 5.8 and 5.5 respectively. Nifedipine (0.1 microM) produced a rightward shift of the concentration-response curves to both LTC4 and LTD4 and depressed their maximal responses. An unacceptably high level of non-specific binding of [3H]LTD4 to membrane preparations of ferret spleen prevented characterisation of this receptor by ligand binding. These results suggest that the ferret spleen has a homogeneous population of a PL receptor type which is insensitive to existing PL1 receptor antagonists. The functional characteristics of this PL receptor type are similar to those of the PL2 receptor on other tissues. The absence of PL1 receptors on this tissue makes it particularly useful in identifying new and selective drug tools for the PL2 receptor.

A second cysteinyl leukotriene receptor in human lung.

Leukotrienes (LT) are potent spasmogenic agents in human isolated bronchial and pulmonary venous muscle preparations. Treatment of human isolated pulmonary veins with the L-serine borate complex (45 mM; 30 min) did not alter the LTC4 pD2 values in these preparations. The cysteinyl LT antagonists, ICI 198615, MK 571 and SKF 104353, significantly shifted to the right the LT concentration-effect curves in airways with pKB values against LTC4 of 8.4 for ICI 198615, 8.6 for MK 571 and 8.0 for SKF 104353. Similar results were found against LTD4. In contrast, these antagonists did not inhibit the LTC4 and LTD4 contractions in human pulmonary veins. LTE4 was a partial agonist on the human pulmonary veins and blocked the contractions with a pKp value of 6.3 against LTD4 and 6.6 against LTC4. An LT analog, BAY u9773, also blocked the LT contractions in bronchial and venous muscle preparations with pKp values against LTD4 and LTC4 of 6.5 and 6.7, respectively. These data provide pharmacological evidence for a second cysteinyl LT receptor in the human lung. One LT receptor (LT-1) is stimulated by all cysteinyl LT, found on airways and inhibited by the LT-1 antagonists, and a second receptor (LT-2) can also be stimulated by all cysteinyl LT and is found on pulmonary veins, resistant to LT-1 antagonists but blocked by LTE4 and the dual LT-1/LT-2 antagonist BAY u9773.

Evidence for two leukotriene receptor types in the guinea-pig isolated ileum.

Leukotriene (LT) receptors in the guinea-pig ileum were characterized using LTB4, LTC4, LTD4 and LTE4 and the LT antagonists FPL 55712, ICI 198615 and (+/-)SKF 104353. LTB4 was inactive but the other LTs induced concentration-related contractions. LTC4 responses differed to those induced by LTD4 or LTE4. Inhibitors of LT metabolism had no significant effects on any LT responses. LTD4 contractions were inhibited by all three antagonists but a resistant response was apparent at concentrations of ICI 198615 greater than 10(-8) M. All three antagonists were weak/inactive against LTC4. LTE4 was a partial agonist which antagonized LTD4 responses but had little or no activity against LTC4 or histamine. These results suggest that two distinct LT receptor types exist on guinea-pig ileum. One type is predominantly activated by LTD4 and is antagonized by three structurally distinct LT antagonists and the partial agonist LTE4. The second type is predominantly activated by LTC4 and is insensitive to the LT antagonists.

The inhibition of [3H]leukotriene D4 binding to guinea-pig lung membranes. The correlation of binding affinity with activity on the guinea-pig ileum.

Guinea-pig lung membranes contain high affinity (KD = 0.8 nM) binding sites for [3H]leukotriene D4 [( 3H]LTD4). The binding is inhibited by leukotriene antagonists, such as ICI 198615 and SK&F 104353, in a manner consistent with the Law of Mass Action at a single site. It is also inhibited by a range of leukotriene analogues in a dose-related manner. Inhibition by some of these e.g. LTC4 suggests that the [3H]LTD4 binding sites are heterogeneous. The binding affinity of the leukotriene analogues is significantly correlated (P less than 0.001) to their spasmogenic activity on guinea-pig ileum but not on guinea-pig lung strip. The binding affinity of the leukotriene antagonists is also correlated to their antagonist activity on guinea-pig ileum (P less than 0.05) but not on guinea-pig lung. These results indicate that the [3H]LTD4 binding site in guinea-pig lung is similar to the leukotriene receptor activated by LTD4 and LTE4 on guinea-pig ileum. The contractile response of guinea-pig lung strips to leukotrienes, in the presence of indomethacin, is mediated by a distinct type of leukotriene receptor.

Autoradiographic localization of leukotriene C4 and D4 binding sites in guinea-pig lung.

Binding of [3H]leukotriene C4 and D4 to guinea-pig lung sections was characterised and binding sites were localized by autoradiography. Both leukotrienes bound to guinea-pig lung sections and membranes with high affinity and with similar characteristics to binding in a membrane preparation. Autoradiography revealed that the distribution of LTC4 and D4 binding sites was markedly different. Smooth muscle and epithelium of central and peripheral airways were densely labelled with [3H]LTC4; vascular smooth muscle and alveolar walls were also labelled. With [3H]LTD4, however, there was no detectable labelling of airways or vessels but substantial labelling of alveolar walls. This lends further support that LTC4 and LTD4 binding sites differ and may not be identical with functional receptors.

The binding of [3H]leukotriene C4 to guinea-pig lung membranes. The lack of correlation of LTC4 functional activity with binding affinity.

High affinity binding sites for [3H]leukotriene C4 ([3H]LTC4) have been identified and characterised in guinea-pig lung membranes. [3H]LTC4 bound to these membranes with a pharmacological specificity totally distinct to that previously observed for [3H]LTD4 binding in guinea-pig lung. Scatchard analysis of saturation binding data showed a single class of binding sites, with a dissociation constant (KD) of 52.6 +/- 4.9 nM and a density (Bmax) of 30 +/- 12 pmol/mg membrane protein. The binding was inhibited with high affinity by a variety of glutathione-containing leukotriene analogues. Most notable was the inhibition by 1,2,3,4-tetranor LTC4 (Ki = 118 nM) and S-decylglutathione (Ki = 154 nM) since neither of these compounds were contractile agonists on guinea-pig parenchymal strips or guinea-pig ileum nor were they antagonists of LTC4-induced contractions of these smooth muscle preparations. These results indicate that the observed binding of [3H]LTC4 to guinea-pig lung membranes is not to a contractile receptor.

Comparison of cardiac and hemodynamic effects of platelet-activating factor-acether and leukotriene D4 in anesthetized dogs.

In anesthetized dogs, platelet-activating factor-acether (PAF; 0.2-1.6 micrograms/kg) and leukotriene (LT) D4 (0.5, 1, and 3 micrograms/kg) were injected into the left circumflex (LCX) coronary artery. Cardiac and systemic hemodynamics, and the ECG were continuously recorded. PAF reduced cardiac performance and affected hemodynamics in a dose-dependent manner: At 7 +/- 3s, LCX flow initially increased by 40%-172% followed by a reduction of 43%-100%, and coronary diameter (measured with ultrasonic techniques) decreased by 4%-10%. Total and late coronary resistance increased. Left ventricular (LV) systolic pressure fell by 22%-48% and LV filling pressure decreased by 5 mm Hg after 0.8 microgram/kg PAF. The LVdP/dtmax diminished by 38%-47%. Peak blood pressure reduction (35%) occurred 60 s after PAF application and lasted for 1.4 min. Heart rate decreased by 10%-17% at peak PAF actions. LTD4 reduced LCX flow by 38%-87%, and coronary diameter by 5%-12%, returning to control value within 3.4 min. Blood pressure, LV pressure, and LVdP/dtmax decreased while heart rate and LV filling pressure increased. ST segments and R-wave voltage of the ECG in lead II elevated after either compound although the effects were more pronounced after LTD4. Indomethacin (5 mg/kg i.v.) pretreatment did not affect LTD4 actions on cardiohemodynamics, but the putative leukotriene antagonist FPL 55712 (1 mg/kg i.v.) blocked LTD4 actions on the heart and circulation. PAF influences on LCX flow were modified by indomethacin: initial flow rose by 250%, and coronary diameter fell by 12%, followed by sustained flow and diameter reduction during the second phase on PAF action. FPL 55712 did not affect the early flow increase after PAF but attenuated the later flow reduction, which was blocked by indomethacin. Thus, PAF and LTD4 may have effects on canine conduit arteries besides their effects on the coronary resistance vessels. The circulatory derangement after PAF may be aggravated by additional eicosanoid release. PAF and LTD4 may be involved in coronary blood flow variations and negative inotropy accompanying anaphylactic disease state.

Effects of intracoronary leukotriene D4 infusion on the coronary circulation, hemodynamics, and prostanoid release in porcine experiments.

The effects of leukotriene (LT) D4 (0.5 microgram/min) infusion into the left anterior descending coronary artery (LAD) of anesthetized pigs were studied in the absence or presence of indomethacin (5 mg/kg i.v.). In some pigs, arteriocoronary venous (A-V) blood plasma concentrations of thromboxane B2 and 6-keto-prostaglandin F1 alpha were measured by RIA technique during LTD4 actions. LTD4 changed LAD blood flow at 5 and 30 s, 2, 4 and 5 min by -46% (p less than 0.01), -93% (p less than 0.001), -45% (p less than 0.01), -28% (p less than 0.05) and 16%, respectively. Thus, the coronary constriction disappeared despite sustained LTD4 infusion and was followed by reactive hyperemia. Indomethacin did not affect the coronary flow or hemodynamic responses to LTD4 infusion. The A-V difference in prostanoids did not change during LTD4 administration. Cyclooxygenase products of arachidonic acid may not be responsible for early constriction and later escape phases of LTD4 effects on the coronary circulation. However, intracoronary bolus injection of LTD4 stimulated prostacyclin release from the vasculature in a dose-dependent manner causing significant reactive hyperemia after temporary blood flow cessation. Release of a LTD4-induced vasodilating factor from the coronary vessels unrelated to prostanoids may cause escape from vasoconstriction during LTD4 infusion.

Leukotrienes on porcine hemodynamics and prostanoid release.

We studied the effect of intracoronary leukotriene B4, C4, D4 and E4 (0.1-3 micrograms) on coronary artery blood flow and resistance in anesthetized pigs. Conventional hemodynamics were measured, and the peripheral electrocardiogram was obtained in lead II. Thromboxane B2 and 6-keto-prostaglandin F1 alpha (as breakdown products of thromboxane and prostacyclin, respectively) were measured during the influence of leukotrienes on the heart. All leukotrienes except B4 reduced coronary flow. Peak reduction was produced by 3 micrograms of each eicosanoid: C4 = 96 +/- 4%+; D4 = 98 +/- 2%+; E4 = 82 +/- 8%+. Coronary resistance increased after the same dose B4 = 65 +/- 18%; C4 = 225 +/- 94% (P less than 0.01); D4 = 442 +/- 118%+; E4 = 110 +/- 43% (+ = P less than 0.001). Increase in filling pressure and heart rate but blood pressure reduction and diminution in left ventricular d P/dtmax were observed with leukotriene C4, D4 and E4. The S-T segments of the electrocardiogram were elevated, thus indicating myocardial ischemia during the blood flow reduction. Indomethacin (5 mg/kg i.v.) had no effects on the leukotriene-induced hemodynamic sequelae. Thromboxane B2 concentration in coronary sinus blood plasma increased by 132-176% (P less than 0.05) at peak leukotriene effects on blood flow. Thus, leukotriene C4, D4, and E4 are vasoconstrictors in the situ porcine heart. Leukotriene B4, however, exerts no hemodynamic effects. The electrocardiographic ischemia and changes in hemodynamics indicate actions on coronary resistance and myocardial depression. These eicosanoids may contribute to cardiac dysfunction and vasospasm in coronary artery disease.

Molsidomine on cardiovascular leukotriene D4 actions.

Anaesthetized open-chest dogs were used to study the effects of intracoronary leukotriene D4 (LTD4; 0.5 micrograms/kg) on haemodynamics, the electrocardiogram (ECG), coronary blood flow in the left circumflex artery and coronary resistance in the absence or presence of the antianginal drug molsidomine (500 micrograms/kg i.v.). LTD4 increased left ventricular (LV) filling pressure from 6.5 +/- 3.8 to 14.7 +/- 3.2 mm Hg (P less than 0.01), decreased LV dP/dtmax from 2,500 +/- 200 to 1,240 +/- 205 mm Hg/s (P less than 0.05), elevated the S-T segment of the ECG from 0.3 +/- 0.2 to 2.4 +/- 0.6 mV (P less than 0.01), and coronary resistance from 3.4 +/- 0.9 to 33.7 +/- 4.8 mm Hg X min X ml-1 (P less than 0.001). Coronary artery blood flow fell from 33.8 +/- 2.7 to 3 +/- 3 ml/min (P less than 0.001). Molsidomine treatment 15 min prior to repeated intracoronary LTD4 application attenuated vasoconstrictor response to LTD4 and the subsequent elevation in total coronary vascular resistance. The negative inotropic actions of the eicosanoid were counteracted by molsidomine. Filling pressure decreased and no temporary signs of ischaemia were noted in the ECG. Inhibition of the cyclooxygenase enzyme activity by i.v. indomethacin (5 mg/kg) had no effects on the LTD4-induced haemodynamic alterations, the ECG, and coronary flow and resistance. The antagonistic molsidomine actions on haemodynamic and electrocardiographic LTD4 effects were not influenced by previous indomethacin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Nifedipine on cardiovascular leukotriene D4 actions in the anaesthetized dog.

Open-chest dogs were used to study the effects of intracoronary leukotriene (LTD4; 0.5 microgram/kg) on haemodynamics, coronary blood flow in the left circumflex artery and coronary resistance in the absence or presence of nifedipine (10 micrograms/kg, i.v.). LTD4 increased ventricular filling pressure by 133% (P less than 0.001), the S-T segments and coronary resistance by 490% (P less than 0.001) and abolished coronary flow for 3 min. Nifedipine pretreatment inhibited the cessation of coronary flow. No change in filling pressure and no ischemia signs in the peripheral ECG were noted. However, nifedipine did not inhibit the LTD4-induced decrease in left ventricular dP/dtmax. Haemodynamic LTD4 actions thus could be partially reversed by i.v. nifedipine, suggesting that leukotriene effects on the coronary and peripheral circulation are mediated by calcium release.