Metabolic cooperation between vascular endothelial cells and smooth muscle cells in co-culture: changes in low density lipoprotein metabolism.

A microcarrier co-culture system for aortic endothelial cells and smooth muscle cells (SMCs) was developed as a model for metabolic interactions between cells of the vessel wall. Low density lipoprotein (LDL) metabolism in SMCs was significantly influenced by co-culture with endothelium. The numbers of high affinity receptors for LDL was increased more than twofold (range, 2.1-5.6), with concomitant increases in LDL receptor-mediated endocytosis and degradation. These effects reached a plateau at an endothelial cell/SMC ratio of 1. Kinetic analysis of the endocytic pathway for LDL in SMCs indicated that, in co-culture with endothelium, there was no alteration in the binding affinity of LDL to its receptors but that the internalization rate constant declined and the rate constant for degradation increased. This analysis suggested that the formation and migration of endocytic vesicles was the rate-limiting step of enhanced LDL metabolism under co-culture conditions. Two mechanisms by which endothelial cells influenced smooth muscle LDL metabolism were identified. First, mitogen(s) derived from endothelial cells stimulated entry of SMCs into the growth cycle, and the changes in LDL metabolism occurred as a consequence of G1-S transition. Second, SMC lipoprotein metabolism was stimulated in the absence of mitogens by a low molecular weight (less than 3,500) factor or factors. Co-culture was a required condition for the latter effect, suggesting that the mediator(s) may be unstable or that cell-cell communication was necessary for expression. These results (a) demonstrate that vascular cell interactions can modify LDL metabolism in SMCs, (b) provide some insights into the mechanisms responsible, and (c) identify co-culture as an experimental approach appropriate to certain aspects of vascular cell biology.

Further subclassification of ATP receptors based on agonist studies.

Recent studies using agonist analogues of ATP and other nucleotides have generated some surprising observations which may have ramifications for the classification of P2 receptors, particularly for those responses currently attributed to P2Y receptor activation. 2-MethylthioATP (2-MeSATP), the conventional P2Y receptor agonist, does not interact with ATP in the expected fashion in various models of endothelial function, suggesting that it acts by a different mechanism. Furthermore, in certain cell types where responses to ATP are mediated by phospholipase C activation, 2-MeSATP has little or no activity. Interestingly, the pyrimidine uridine triphosphate (UTP) invariably shows similar potency to ATP in systems where 2-MeSATP is inactive. In this article Steve O'Connor and colleagues discuss these data and their significance, and propose that separate receptors may be responsible: one sensitive to 2-MeSATP and the other, a 'nucleotide' receptor, sensitive to UTP.

Effect of N-linked glycosylation on glycopeptide and glycoprotein structure.

Asparagine-linked glycosylation is an enzyme-catalyzed, co-translational protein modification reaction that has the capacity to influence either the protein folding process or the stability of the native glycoprotein conjugate. Advances in both glycoconjugate chemical synthesis and glycoprotein expression methods have increased the availability of these once elusive biopolymers. The application of spectroscopic methods to these proteins has begun to illuminate the various ways in which the saccharide affects the structure, function and stability of the proteins.

Modulation of protein structure and function by asparagine-linked glycosylation.

In eukaryotic cells, many enzymes are devoted to the construction of the complex glycan structures that decorate secreted and cell-surface proteins. Recent studies have begun to elucidate the effects of asparagine-linked glycosylation on protein folding and on the structure and function of mature glycoproteins.

Role of distinct subpopulations of peritoneal macrophages in the regulation of reactive oxygen species release.

It has been reported in vitro that during the respiratory burst of phagocytic cells the superoxide anion production per cell shows a negative relation with the cell density. This process has been described as autoregulation. The aim of this work was to analyze the superoxide anion production in thioglycollate-elicited peritoneal macrophage exudates to evaluate the importance of the peritoneal cavity environment in the autoregulation process. 12-O-tetradecanoylphorbol-13-acetate (PMA) was used to stimulate the respiratory burst and superoxide anion production was measured evaluating the intracellular formazan deposits that precipitate as a result of nitro blue tetrazolium (NBT) reduction. We have demonstrated a negative correlation between superoxide anion production and cell density in the peritoneal cavity in macrophages challenged with PMA. The response of individual cells was analyzed by means of an image analyzer, measuring the amount of formazan per cell and cell-size changes during the process of activation. The results revealed that the decrease in individual cell response as a function of higher cell densities were due to a significant increase in the amount of basal reaction macrophages. Concomitantly, the number of reactive cells remained unchanged irrespective of the cell density of the population. A direct correlation between cell size and superoxide anion production was observed. This phenomenon was demonstrated in SENCAR and Balb/c strains. However, macrophages from SENCAR mice showed greater superoxide anion production than those from Balb/c. The differences between strains could be associated to the increased sensitivity to PMA tumor promotion of SENCAR mice. Based on this property, macrophages from SENCAR mice were stimulated with opsonized zymosan, a particulate stimulus that reflects the interaction macrophage-microorganism during the phagocytic process. This data will contribute to the knowledge of infection control. We conclude that variations in basal reaction cells modulates the macrophage activation response when excess macrophages are recruited to the peritoneum. This is demonstrated using different stimuli, thus suggesting that this response may be applied to a wide variety of stimuli-macrophage interactions. The differences between strains may be associated to the increased sensitivity to PMA tumor promotion of SENCAR mice.

SSR182289A enhances thrombolysis induced by fibrinolytic agents in rabbit models of venous and arterial thrombosis.

BACKGROUND AND OBJECTIVES: The purpose of this work was to investigate whether thrombolysis induced by recombinant tissue plasminogen activator (rt-PA) or streptokinase (SK) was enhanced in different rabbit models of thrombolysis by SSR182289A, a novel synthetic direct thrombin inhibitor which has been shown to possess potent antithrombotic properties in several experimental animal models. METHODS AND RESULTS: Human rt-PA alone (0.125 mg kg(-1) h(-1) for 2 h) induced a significant thrombolysis (18%, P < 0.05) of a venous-type thrombus in the rabbit jugular vein. Under these conditions, SSR182289A (3 mg kg(-1) i.v. bolus) inhibited 125I-fibrin accretion onto a preformed thrombus in the rabbit jugular vein by 72%, but was unable to induce thrombolysis on its own. However, coadministration of SSR182289A and rt-PA strongly enhanced rt-PA-induced thrombolysis (38.4%, P < 0.01). The effect of SSR182289A was further assessed in a model of thrombolysis of an electrical injury-induced, stable (occlusion duration > 2 h) thrombus formed in the rabbit femoral artery. Whereas local intra-arterial infusion of high doses of SSR182289A (3 mg kg(-1) h(-1) for 1 h) alone was able to restore flow, SK (12,000 U kg(-1) h(-1)) and a low dose of SSR182289A (0.3 mg kg(-1) h(-1)) were ineffective. However, intra-arterial coadministration of SSR182289A (0.3 mg kg(-1) h(-1)) and SK (12,000 U kg(-1) h(-1)) induced significant reflow (time to reflow was shortened by 34.7 +/- 7.5 min, P < 0.05). In the same model, systemic i.v. administration of high doses of SSR182289A (10 mg kg(-1) i.v. bolus) and rt-PA (1 mg kg(-1) h(-1)) alone did not induce any thrombolysis. However, the association of both compounds quickly (30 +/- 6 min) restored and maintained flow (duration > 2 h) in all animals. CONCLUSIONS: The present results show that bolus i.v. injection of SSR182289A is able to potentiate thrombolysis induced by two fibrinolytic agents whether the thrombus is of venous or arterial origin, thus suggesting that SSR182289A may be of use as an adjunct to thrombolysis.

Anticoagulant activity and pharmacokinetic properties of a sub-cutaneously administered mixed micellar formulation of argatroban in experimental animals.

We have studied the anticoagulant properties of a novel mixed micellar formulation containing 14 mg/ml argatroban administered by the sub-cutaneous (s.c.) route to rats, rabbits, dogs and primates. Blood samples were taken at various times post-treatment for the determination of the thrombin time (TT), Ecarin clotting time (ECT) and the activated partial thromboplastin time (aPTT). Plasma levels of argatroban were determined in the dog and primate. Mixed micelles alone (0.15 M sodium glycocholate and 0.15 M egg lecithin) were without effect on the clotting parameters. The mixed micellar formulation of argatroban dose-dependently increased all three clotting parameters in the rat (1-4 mg/kg), the rabbit (1 and 2 mg/kg), the dog (1 and 2 mg/kg) and the primate (0.25 and 0.5 mg/kg). In each case the TT was the most sensitive parameter, followed by the ECT and the aPTT. The duration of action of argatroban in each species was dose dependent and varied from 3 h in the rat to 6 h in the dog. In the latter, the mixed micelle formulation had a significantly increased plasma half-life and mean residence time without affecting the overall area under the curve. The increases in the clotting time were strongly correlated with the plasma levels of argatroban and were linear across the range of concentrations obtained in the dog and the primate, although the aPTT plasma concentration response curve was very flat. Species differences were noted between the increase in clotting time for a given plasma concentration, with the primate being more sensitive than the dog (e.g. 4.7 times more so in terms of the ECT). Thus, a mixed micellar formulation of argatroban, which markedly enhances its solubility, could be useful as a potential anticoagulant for sub-cutaneous administration.

Antiplatelet and antithrombotic activity of SL65.0472, a mixed 5-HT1B/5-HT2A receptor antagonist.

The antiplatelet and antithrombotic activity of SL65.0472 (7-fluoro-2-oxo-4-[2-[4-(thieno [3,2-c]pyrin-4-yl) piperazin-1-yl]ethyl]-1,2-di-hydroquinoline-acetamide), a mixed 5-HT1B/5-HT2A receptor antagonist was investigated on 5HT-induced human platelet activation in vitro, and in rat, rabbit and canine platelet dependent thrombosis models. SL65.0472 inhibited 5-HT-induced platelet shape change in the presence of EDTA (IC50 values = 35, 69 and 225 nM in rabbit, rat and human platelet rich plasma (PRP)), and also inhibited aggregation induced in human PRP by 3-5 microM 5-HT + threshold concentrations of ADP (0.5-1 microM) or collagen (0.3 microg/ml) with mean IC50 values of 49 +/- 13 and 48 +/- 6 nM respectively. SL65.0472 inhibited thrombus formation when given both intravenously 5 min and orally 2 h prior to assembly of an arterio-venous (A-V) shunt in rats as from 0.1 and 0.3 mg/kg respectively. It was active in a rabbit A-V shunt model with significant decreases in thrombus weight as from 0.1 mg/kg i. v. and at 10 mg/kg p.o. The delay to occlusion in an electric current-induced rabbit femoral artery thrombosis model was increased by 251% (p <0.05) after 20 mg/kg p.o. SL65.0472 (30 microg/kg i.v.) virtually abolished coronary cyclic flow variations (7.2 +/- 1.0/h to 0.6 +/- 0.6/h, p <0.05) and increased minimum coronary blood flow (1.2 +/- 0.8 ml/min to 31.8 +/- 8.4 ml/min, p <0.05) in a coronary artery thrombosis model in the anaesthetised dog. Finally, SL65.0472 significantly increased the amount of blood lost after rat tail transection at 3 mg/kg p.o. Thus the anti-5-HT2A component of SL65.0472 is reflected by its ability to inhibit 5-HT-induced platelet activation, and platelet-rich thrombus formation.

An introduction to the pharmacologic properties of Dopacard (dopexamine hydrochloride).

Dopexamine hydrochloride (Dopacard) has been developed as a peripherally acting dopamine receptor agonist with afterload reducing properties for use in the acute management of low cardiac output states. Dopexamine hydrochloride is one-third as potent as dopamine in stimulating DA1 receptors but 60 times as potent as a beta 2-adrenoceptor agonist. Unlike dopamine, it is a weak beta 1-adrenoceptor agonist and does not stimulate vascular alpha adrenoceptors. Its stimulant properties at vascular DA1 receptors and at vascular beta 2 adrenoceptors endow it with the ability to improve renal blood flow and to increase cardiac output secondary to afterload reduction. In addition, mild positive inotropic activity arises from stimulation of cardiac beta 2 adrenoceptors, potentiation of endogenous norepinephrine due to uptake-1 blockade, and activation of the baroreceptor reflex. Other features of dopexamine hydrochloride that should enhance its clinical use are lack of arrhythmogenicity and rapid responsiveness to alterations in infusion rate.

Presence of vasoconstrictor 5HT1-like receptors revealed by precontraction of rabbit isolated mesenteric artery.

1. A series of 5-hydroxytryptamine (5-HT) receptor agonists including 5-HT, 5-carboxamidotryptamine (5-CT) and sumatriptan produced little or no contraction of rabbit isolated mesenteric arteries under resting tone conditions, even at concentrations up to 10(-4) M. 2. When the same agonists were retested in mesenteric artery preparations pre-contracted with the thromboxane-mimetic, U46619, each demonstrated concentration-related vasoconstrictor activity. 5-CT and 5-HT were especially potent and effective in this model giving EC50 values of 4.3 x 10(-9) M and 1.6 x 10(-8) M respectively and maximum effects equivalent to those of KCl 80 mM. In preparations precontracted by U46619 (conditions retained throughout the rest of the study) the order of agonist potency was 5-CT > 5-HT > RU 24969 = sumatriptan > 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT) > cisapride. 3. The vasoconstrictor effects of 5-CT were competitively antagonized by methiothepin (pA2 8.20) but resistant to antagonism by a range of other 5-HT receptor antagonists, i.e. pindolol (5-HT1A/5-HT1B), propranolol (5-HT1B), spiperone (5-HT2A), ondansetron (5-HT3), ICS 205930 (5-HT3/5-HT4) and SDZ 205557 (5-HT4). 5-CT responses were slightly antagonized by a high concentration of ritanserin (5-HT2A/5-HT2C). Responses to 5-HT and sumatriptan were also antagonized by methiothepin with similar affinity (pA2/pKB values congruent to 8.0). 4. Metergoline and rauwolscine (10(-7)-10(-6) M) antagonized the effects of 5-CT in a non-competitive fashion giving pKBapp values of 7.13 (metergoline) and 6.86 (rauwolscine). 5. Vasoconstrictor responses to 5-HT were not modified in the presence of ritanserin (3 x 10-7 M) orspiperone (3 x 10-7 M) and only modestly antagonized by ketanserin (10-6 M) suggesting that 5-HT2Areceptors do not make a significant contribution in this model.6. Hence, precontraction of rabbit mesenteric arteries reveals potent vasoconstrictor effects of 5-HT and related agonists. Based on the agonist potency order and the antagonist studies performed, the receptor subtype responsible has the characteristics of a 5-HT1-like (probably 5-HTlD) receptor. This study therefore demonstrates a particularly striking example of vasoconstrictor synergy involving 5-HT1-like receptors.

Sumatriptan-induced saphenous venoconstriction in the anaesthetized dog through 5-HT1-like receptor activation.

1. The role of vasoconstrictor 5-HT1-like receptors in the control of vascular reactivity in vivo has been relatively little studied, particularly with regards to venous function. Using an anaesthetized dog model, we have investigated the haemodynamic profile of the selective 5-HT1-like agonist, sumatriptan, focussing on the reactivity of the saphenous venous bed. The key feature of our experimental model was the implantation of ultrasonic crystals on the adventitial surface of the lateral saphenous vein to provide direct and continuous measurement of drug-induced changes in vein diameter. Saphenous vein pressure was measured simultaneously via a proximal branch. 2. Sumatriptan 1-30 micrograms kg-1, i.v., produced pronounced dose-related reductions in saphenous vein diameter which reached congruent to 40% at the highest dose tested. Sumatriptan also produced modest increases in mean blood pressure, total peripheral resistance and left ventricular end diastolic pressure but had little or no effect on cardiac output, heart rate, cardiac contractility or saphenous venous pressure. Sumatriptan-induced reductions in saphenous vein diameter were strongly antagonized by the 5-HT1-receptor antagonist, methiothepin (0.3 mg kg-1, i.v.) but were unaffected by the 5-HT2 antagonist, ketanserin (0.3 mg kg-1, i.v.). 3. Hence, 5-HT1-like receptor stimulation in vivo can result in a powerful local venoconstrictor effect.

Quantitative analysis of the agonist and antagonist actions of some ATP analogues at P2X-purinoceptors in the rabbit ear artery.

1. The agonist and antagonist effects of a series of beta, gamma-methylene dihalo- and 2-methylthio-substituted analogues of ATP at P2x-purinoceptors have been analysed on the rabbit isolated ear artery preparation. Cumulative and sequential dosing experimental protocols were employed in the construction of agonist concentration-effect curves in order to address the possible influence of acute receptor desensitization on subsequent analyses. 2. Using the cumulative curve design the following results were obtained: D-AMP-PCBr2P, 2-methylthio-D-AMP-PCCl2P, L-AMP-PCF2P, L-AMP-PCCl2P and LAMP-PCBr2P each behaved as partial agonists. D-AMP-CPP was used as a reference full agonist and these analogues were analysed by the comparative method of Barlow et al. (1967), to provide estimates of affinity and efficacy. 2-Methylthio-L-AMP-PCBr2P was virtually silent as an agonist and was analysed as a competitive antagonist by Schild analysis. 3. Two agonists, L-AMP-PCCl2P and L-AMP-PCBr2P, were analysed by the sequential curve design, and the antagonist effects of one of the agonists, L-AMP-PCBr2P were also analysed using this protocol. The resulting estimates of affinity and efficacy, while similar to those obtained with the cumulative design, indicated that acute desensitization may affect curve definition and estimation of these quantities. 4. The following structure-activity trends emerged: D-analogues tended to have higher efficacy but lower affinity than L-analogues; efficacy varied markedly and inversely with the atomic weight of the halogen while affinity was only minimally affected; 2-methylthio- substitution also reduced efficacy with minimal effect on affinity. 5. The results of this analysis are discussed in terms of the utility of affinity and efficacy information in the classification of purinoceptors and the design of chemical probes for them.

Comparison of the haemodynamic profiles of elgodipine and nicardipine in the anaesthetized dog.

1. The haemodynamic profile of elgodipine (1-30 micrograms kg-1, i.v.), a new dihydropyridine calcium antagonist, has been compared directly with that of nicardipine (1-30 micrograms kg-1, i.v.) in chloralose-anaesthetized dogs. 2. Nicardipine produced dose-related systemic, pulmonary and coronary vasodilatation accompanied by reflex tachycardia, inotropy and increases in cardiac output and myocardial oxygen consumption (MVO2). Elgodipine had similar vasodilator and hypotensive properties to nicardipine but produced less reflex inotropy, little or no reflex tachycardia and did not increase MVO2. 3. Both calcium antagonists were retested in a separate group of anaesthetized dogs pretreated with propranolol (1 mg kg-1, i.v.) and atropine (0.3 mg kg-1, i.v.) to abolish reflex autonomic tone to the heart and thus reveal the direct cardiac effects of each compound. Under these conditions both elgodipine and nicardipine decreased heart rate and cardiac contractility and slowed atrio-ventricular conduction. Elgodipine was approximately ten times more potent than nicardipine as a decelerator agent and slightly more potent in depressing cardiac contractility and increasing PR interval duration. Elgodipine, unlike nicardipine, slightly reduced the QTc interval of the electrocardiogram. Therefore, the potent decelerator effect of elgodipine, which was present throughout the dose-range, appears to be largely responsible for the suppression of reflex tachycardia observed when the baroreflex is functional. 4. Elgodipine is a potent systemic and coronary vasodilator with more marked direct cardiac effects than nicardipine, particularly with respect to slowing of heart rate. The ability of elgodipine to increase coronary blood flow without significant reflex tachycardia or increases in MVO2 suggests that this compound will have a more favourable effect on myocardial oxygen supply/demand balance than nicardipine. The haemodynamic profile of elgodipine may be suitable for the treatment of angina.

Characterization of P2x-receptors in rabbit isolated ear artery.

1. The isolated central ear artery of the rabbit contracts in response to adenosine 5'-triphosphate (ATP) and analogues, effects proposed to be mediated by stimulation of P2x-receptors. We have extended the characterization of the purinoceptor in this tissue by examining the effects of a series of receptor agonists. The study was designed in such a way as to avoid factors which normally limit attempts to classify receptors on the basis of agonist potency orders. 2. D-alpha, beta-methylene ATP (D-alpha, beta-meATP), D-beta, gamma-methylene ATP (D-beta, gamma-meATP), L-beta, gamma-methylene ATP (L-beta, gamma-meATP), 2-methylthio-D-ATP (2-MeSATP) and ATP produced concentration-related contractions of the ear artery with similar maximum responses, suggesting that they were full agonists. Selective desensitization of P2x-receptors abolished or greatly reduced responses to D-alpha, beta-meATP, L-beta, gamma-meATP, D-beta, gamma-meATP. Responses to ATP were inhibited by by desensitization but a significant resistant component was still apparent. 3. D-alpha, beta-meATP was the most potent agonist tested (pA50 6.47 +/- 0.04) being 2138 times more potent than ATP and approximately 9 times more potent than L-beta, gamma-meATP. The agonist potency order was: D-alpha, beta-meATP greater than L-beta, gamma-meATP greater than D-beta, gamma-meATP greater than or equal to 2-MeSATP greater than ATP. This is generally consistent with the order proposed for P2x-receptors. The relative potencies of P2x-agonists in the rabbit ear artery show both similarities to and differences from data obtained in other smooth muscle preparations.

Suramin is a slowly-equilibrating but competitive antagonist at P2x-receptors in the rabbit isolated ear artery.

1. The antagonist dynamics of suramin were investigated at P2x-receptors in isolated rings of endothelium-denuded ear artery from New Zealand White (NZW) rabbits. 2. alpha, beta-Methylene adenosine 5'-triphosphate (ATP) concentration-effect curves were constructed cumulatively in a paired curve design in the absence and presence of increasing concentrations of suramin, incubated for 45 min. The slope of the resulting Schild plot was significantly greater than unity (1.50 +/- 0.08). 3. Assuming that slow equilibration by suramin explains the steep Schild plot, further experiments were conducted using short (15 min) and long (3 h) incubation times. The resulting Schild plot slopes were 1.66 +/- 0.36 and 1.06 +/- 0.13 respectively confirming the assumption. However, after 3 h incubation, suramin also caused depression of alpha, beta-methylene ATP curves. 4. In an attempt to minimize the depressant effect of suramin, a kinetic study was designed to calculate the minimum incubation times for each concentration of suramin used in the Schild analysis to achieve effectively complete equilibrium. Theoretically fractional occupancy for the antagonist is given by (r - 1)/r, where r is the dose-ratio. A plot of (r - 1)/r against time allowed the apparent 'on' and 'off' rate constants to be calculated. 5. With the resulting rate constant estimates, an optimised antagonism study was carried out in which incubation times were chosen such that greater than 95% occupancy by suramin could be achieved without agonist curve depression at each concentration of suramin used. 6. Under these conditions, suramin fulfilled all criteria for simple competition: parallel rightward displacement of alpha,beta-methylene ATP curves and a Schild plot slope of unity (1.00 + 0.09). The resulting pKB estimate was 4.79 + 0.05. This estimate of affinity was shown to be independent of the agonist used in another experiment in which L-beta-methylene ATP was employed (pKB = 5.17). 7. Under the same conditions, suramin was found to have no effect on KCI-induced contractions and only slight effects on phenylephrine- and histamine-induced responses.8. This analysis provides the first evidence that suramin is a genuine competitive P21-receptor antagonist.

Coronary vasoconstriction in the conscious rabbit following intravenous infusion of L-NG-nitro-arginine.

Intravenous infusion of L-NG-nitro-arginine, an inhibitor of endothelial nitric oxide (NO) synthesis, produced vasoconstriction in the coronary, cerebral, renal and duodenal vascular beds of the conscious rabbit. In this study, using radiolabelled microspheres, we provide in vivo evidence for a basal NO-dependent vasodilator tone in the coronary vascular bed.

Antithrombotic activity of a monoclonal antibody inducing the substrate form of plasminogen activator inhibitor type 1 in rat models of venous and arterial thrombosis.

1. Elevated plasminogen activator inhibitor 1 (PAI-1) is a risk factor for thrombosis, and inhibitors of the interaction between PAI-1 and tissue plasminogen activator (t-PA) have antithrombotic and prothrombolytic activity in animals. We describe the antithrombotic effects in the rat of a monoclonal antibody (MA33H1) which converts PAI-1 to a non-inhibitory substrate. 2. The activity of MA33H1 against rat PAI-1 was confirmed using two-chain t-PA and a chromogenic substrate. MA33H1 was evaluated in rat venous (thromboplastin + stasis in the abdominal vena cava) and arterial (electric current applied to a carotid artery) thrombosis models. The effects on tail-transection bleeding time were studied. 3. MA33H1 at 100 ng ml(-1) inhibited both human (44.1%) and rat PAI-1 (49.7%). This effect was concentration-dependent. Its effect on human PAI-1 was not significantly inhibited by 1 microg ml(-1) fibrin or a approximately 7 fold molar excess of vitronectin (1 nM). Inhibition of rat PAI-1 was unchanged by fibrin, but vitronectin reduced inhibition from 0.5 nM. 4. In the venous thrombosis model, MA33H1 significantly reduced thrombus weights by 38 and 58.6% at 50 and 100 microg kg(-1) min(-1) i.v. respectively. This effect was inhibited by tranexamic acid. In the arterial model, MA33H1 significantly increased the delay to occlusive thrombus formation by 58 and 142% at 50 and 100 microg kg(-1) min(-1) i.v., and did not affect bleeding time at 300 microg kg(-1) min(-1) i.v. 5. Thus, a monoclonal antibody which transforms PAI-1 to a t-PA substrate prevents thrombus formation in the rat with no effect on bleeding time at a higher dose.

Pharmacological properties of FPL 63547, a novel inhibitor of angiotensin-converting enzyme.

1. FPL 63547, in its active diacid form, was a potent inhibitor of rabbit lung angiotension converting enzyme (ACE) in vitro (IC50 0.51 nM). 2. In conscious normotensive dogs, FPL 63547 (10-300 micrograms kg-1 i.v.) produced prolonged, dose-related inhibition of plasma ACE activity and angiotensin I pressor responses, without affecting basal blood pressure, heart rate or pressor responses to angiotensin II. 3. In anaesthetized dogs, FPL 63547 diacid (3-300 micrograms kg-1 i.v. cumulatively) produced dose-related increases in cardiac output accompanied by falls in total peripheral resistance indicative of vasodilatation. Mild stimulation of cardiac rate and contractility was also observed. Enalapril diacid had a similar profile. 4. FPL 63547 was a highly effective antihypertensive agent after oral administration to spontaneously hypertensive rats (SHR) pretreated with a diuretic. It lowered systolic blood pressure (SBP) on acute administration over the range 3 X 10(-7)-10(-5) mol kg-1 p.o. (congruent to 0.13-4.5 mg kg-1 p.o.). FPL 63547 was more potent than other ACE inhibitors tested, threshold active doses for lisinopril, enalapril and captopril being 10(-6), 10(-6) and 3 X 10(-5) mol kg-1 p.o., respectively. The antihypertensive effects of FPL 63547, unlike those of enalapril and captopril, were of long duration. 5. The antihypertensive efficacy of FPL 63547 was also observed following chronic oral administration. A dose of 0.5 mg kg-1 day-1 once daily for 23 days produced a sustained reduction of SBP. By the end of the treatment period, SBP was significantly lowered both pre- and post-dose, i.e. effective 24 h control had been achieved. 6. The profile of FPL 63547 is consistent with it being a potent, selective and long-acting ACE inhibitor. As an antihypertensive agent in SHR it compared favourably with other members of this class with respect to potency and duration of action.

Preferential biliary elimination of FPL 63547, a novel inhibitor of angiotensin-converting enzyme, in the rat.

1. The route of elimination of FPL 63547, a novel inhibitor of angiotensin-converting enzyme (ACE), has been investigated in the anaesthetized rat. Comparisons have been made with other ACE inhibitors. 2. Bile and urine samples were collected over a 5 hour period following a single i.v. dose of ACE inhibitor (2 mumol kg-1). Samples were bioassayed for ACE inhibitory activity using affinity-purified rabbit lung ACE and the amounts of the active form of inhibitor present in each sample were calculated by comparison with a standard curve. 3. FPL 63547 was rapidly and extensively excreted as the diacid in the bile but appeared in the urine in negligible amounts. The bile:urine ratio was 21.4:1 indicating a marked preference for the biliary route. A similar elimination profile was observed when the compound was dosed in its active form (FPL 63547 diacid), 87.9% of which was found in the bile over the 5 h collection period, with a bile: urine ratio of 14.6:1. 4. The marked preference of FPL 63547 for biliary elimination was not shared by the other ACE inhibitors tested in this study. Lisinopril demonstrated the opposite pattern, being excreted almost exclusively by the kidney (bile:urine ratio 0.06:1). Enalapril was eliminated in approximately equal amounts in bile and urine (ratio 0.7:1) while spirapril diacid showed a slight preference for the bile (ratio 2.6:1). 5. The physical chemical properties of FPL 63547 diacid may be responsible for its unusual preference for biliary elimination. In particular, the amphipathic character and strong acid functionality of the compound are thought to favour transport into the bile. 6. Elimination by the biliary route will be preferred in patients whose renal function is impaired as a result of disease or age. In such patients the elimination of renally-excreted ACE inhibitors is known to be compromised, resulting in compound accumulation and the need for closer monitoring. Therefore, the elimination profile of FPL 63547, if confirmed in man, may prove to be clinically advantageous.

The effects of dopexamine on the cardiovascular system of the dog.

The cardiovascular effects of dopexamine and dopamine were compared in the anaesthetized and conscious dog by the use of intravenous infusions over the dose range 3 X 10(-9) - 10(-7)mol kg-1 min-1. In the anaesthetized dog, dopexamine produced a dose-related fall in blood pressure due to peripheral vasodilatation and a small rise in heart rate and contractility. By contrast, dopamine did not significantly reduce blood pressure but produced a larger dose-related increase in contractility. At the highest infusion rate (10(-7)mol kg-1 min-1) blood pressure and heart rate were increased by dopamine. Dopexamine dilated the renal and mesenteric vascular beds with a potency similar to that of dopamine. Femoral vascular responses produced by both agents were inconsistent but the highest infusion rate of dopamine did produce vasoconstriction. With the aid of selective receptor antagonists (haloperidol, propranolol and bulbocapnine) the vasodepressor activity of dopexamine was shown to be mediated by stimulation of DA2-, beta- and DA1-receptors. The cardiac stimulation and renal vasodilatation produced by both compounds were due to stimulation of beta-adrenoceptors and DA1-receptors respectively. In the conscious dog, intravenous infusion of dopexamine caused a dose-related fall in blood pressure, renal vasodilatation and an increase in cardiac contractility and heart rate. Dopamine also increased cardiac contractility, and renal blood flow due to renal vasodilatation but without affecting heart rate. At the highest infusion rate, blood pressure was increased. Dopexamine and dopamine produced a similar incidence of panting and repetitive licking at 3 X 10(-8)mol kg-1 min-1 and emesis at 10(-7)mol kg-1 min-1, due to stimulation of dopamine receptors in the chemoreceptor trigger zone. Dopexamine produces a different cardiovascular profile from dopamine in the anaesthetized and conscious dog. Both compounds reduce renal vascular resistance, but in contrast to dopamine, dopexamine reduces afterload and produces only mild inotropic stimulation. These differences reflect contrasting activity at adrenoceptors.