Effects of vinblastine on malondialdehyde formation, serotonin release and aggregation in human platelets

Effects of the microtubular agent vinblastine on human platelet malondialdehyde formation, [14C]serotonin release and aggregation were studied in suspensions of [14C]serotonin-labelled platelets. Vinblastine caused dose-dependent inhibition of malondialdehyde formation and aggregation in platelet suspensions stimulated with thrombin, ADP or palmitaldehyde acetal phosphatidic acid (PGAP). Malondialdehyde formation, aggregation and [14C]serotonin release caused by threshold doses of thrombin were reduced but not abolished by 100 muM vinblastine; 30-100 muM vinblastine abolished ADP- and PGAP-induced malondialdehyde formation and [14C]serotonin released and transformed ADP- and PGAP-induced irreversible aggregation to a diminished reversible response. Arachidonate conversion to malondialdehyde catalysed by human platelet microsomes was inhibited by vinblastine and the cyclooxygenase inhibitors indomethacin and aspirin, but not by salicylate. Vinblastine inhibited the microsome-catalysed formation of malondialdehyde from prostaglandin H2. It is concluded that vinblastine inhibits the thromboxane pathway of arachidonate metabolism in stimulated platelets, consequently inhibiting release and aggregation, and that this effect of vinblastine may be, at least in part, independent of its antimicrotubular actions.     

Arachidonate metabolism, 5-hydroxytryptamine release and aggregation in human platelets activated by palmitaldehyde acetal phosphatidic acid

Palmitaldehyde acetal phosphatidic acid ( PGAP ) caused dose-dependent aggregation of human platelets resuspended in modified Tyrode medium, with a threshold concentration of 0.5-1 microM and an EC50 of 4 microM. Concentrations of PGAP which elicited biphasic irreversible aggregation concomitantly induced formation of 1.02 +/- 0.029 nmol (mean +/- s.e. mean) of malondialdehyde (MDA) per 10(9) platelets and caused release of 58 +/- 2.8% of platelet [14C]-5-hydroxytryptamine ([14C]-5-HT) from prelabelled platelets; no MDA formation or [14C]-5-HT release occurred at lower doses of PGAP which elicited only monophasic reversible aggregation. Adenosine 5'-pyrophosphate (ADP)-induced platelet activation resulted in formation of 0.344 +/- 0.004 nmol of MDA per 10(9) platelets in association with irreversible aggregation and 49.1 +/- 1% release of [14C]-5-HT. Mepacrine, a phospholipase A2 inhibitor, at 2.5 microM reduced PGAP -induced MDA formation and [14C]-5-HT release by the resuspended platelets without affecting irreversible aggregation; higher concentrations of mepacrine abolished all three responses. Chlorpromazine, a calmodulin antagonist, similarly inhibited PGAP -induced MDA formation and irreversible aggregation, and at 100 microM abolished monophasic aggregation. The cyclo-oxygenase inhibitor indomethacin caused a concentration-dependent reduction of PGAP -induced MDA formation by resuspended human platelets without significantly inhibiting [14C]-5-HT release or irreversible aggregation; concentrations (greater than or equal to 1.75 microM) which inhibited MDA formation by more than 94% abolished [14C]-5-HT release, and converted second phase irreversible aggregation to an extensive reversible response. 2-Methylthioadenosine 5'-phosphate (2 methylthio-AMP), an ADP antagonist, inhibited PGAP -induced MDA formation, [14C]-5-HT release and second phase aggregation in the human platelet suspensions in a parallel, concentration-dependent manner; at 9.4 microM 2-methylthio-AMP, both MDA formation and [14C]-5-HT release were abolished and monophasic, reversible aggregation remained. Albumin was required for aggregation of washed human platelets to PGAP . Irreversible PGAP -induced aggregation of washed [14C]-arachidonate-labelled platelets was accompanied by a low net loss of 14C from platelet phospholipids, an equivalent increase in 14C in free fatty acids, and the appearance of 14C in thromboxane (Tx)B2; mepacrine reduced the loss in 14C from phospholipids and inhibited aggregation and formation of [14C]-TxA2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of selective α-adrenoceptor agonists and antagonists with human and rabbit blood platelets

1 The selectivity of α-adrenoceptors mediating the pro-aggregatory response of human and rabbit platelets to adrenaline and the conditions required to permit expression of an aggregatory response to partial agonists at these α-adrenoceptors have been studied.

2 Yohimbine causes effective blockade of the pro-aggregatory responses whereas indoramin and prazosin are ineffective.

3 The clonidine analogue, UK-14304, is nearly as effective as adrenaline in inducing an aggregatory response in human platelets and a pro-aggregatory response in rabbit platelets. Cross-tachyphylaxis between adrenaline and UK-14304 has been demonstrated.

4 Clonidine is a weak agonist for the pro-aggregatory response of rabbit platelets and in some donors for the aggregatory response of human platelets.

5 Methoxamine induces a pro-aggregatory response in human platelets which is blocked by indoramin or prazosin but not by yohimbine. No such response to methoxamine is observed in rabbit platelets.

6 The divalent cation ionophore, A-23187, induces an aggregatory response to clonidine (in platelets from a non-responsive donor), phenylephrine and methoxamine in human platelets and to adrenaline, UK-14304 and clonidine in rabbit platelets. A secretory response to clonidine is also induced by A-23187 in human platelets.

7 The adenylate cyclase inhibitor, SQ-22536, is ineffective in either inducing a response to the α-agonists or potentiating the effect of A-23187.

8 The aggregatory responses to adrenaline and UK-14304 in rabbit platelets and to clonidine in human and rabbit platelets, which can be induced by A-23187, are blocked by yohimbine but not by prazosin or indoramin.

9 From these studies we conclude that the pro-aggregatory responses of human and rabbit platelets to adrenaline are mediated primarily by α₂-adrenoceptors. The presence of α₁-adrenoceptors on human platelets is confirmed but these receptors do not appear to be present on rabbit platelets. The conditions required for expression of an aggregatory response to partial agonists at the human and rabbit platelet α-adrenoceptors implicate an increase in cytosolic Ca²⁺ concentration as a key event in stimulus-response coupling but do not indicate such a role for depression of cyclic adenosine-3′,5′-monophosphate concentration.

     

Interactions of dopamine and the release of [3H]-taurine and [3H]-glycine from the isolated retina of the rat

1 The dose-related, calcium-dependent, potassium-stimulated release of preloaded [³H]-dopamine from the superfused rat retina has been demonstrated.

2 A high-affinity uptake system for dopamine exists in rat retina in vitro; K_m value was calculated as 1.89 μM, V_max value as 1.4 nmol g^-1 tissue h^-1.

3 Dopamine (0.8 and 4 mM) inhibited the spontaneous release of [³H]-glycine from retina, and in the case of 0.8 mM dopamine this inhibitory effect was antagonized by 10 μM (+)-butaclamol but not by 10 μM (-)-butaclamol.

4 The potassium-evoked (25 mM) release of [³H]-glycine from rat retina was similarly inhibited by dopamine (0.4-4 mM) in a dose-related manner when added to the superfusate with the potassium. The effect of 0.8 mM dopamine was antagonized by 10 μM (+)-butaclamol but not by 10 μM (-)-butaclamol.

5 Dopamine (4 mM) significantly reduced the spontaneous release of [³H]-taurine from rat retina.

6 The potassium-stimulated (25 mM) release of [³H]-taurine occurred after the cessation of the depolarizing stimulus. This delayed release of [³H]-taurine was unaffected if dopamine was applied to the superfusate at the same time as the potassium, but it was significantly reduced if dopamine (0.8 and 4 mM) was applied after the depolarizing stimulus had been removed and during the actual amino acid release phase.

7 The inhibition of K⁺-stimulated (25 mM) delayed release of [³H]-taurine by applying dopamine (0.8 mM) after the depolarizing stimulus was blocked by 10 μM (+)-butaclamol but not by 10 μM (-)-butaclamol.

8 The results are discussed with respect to the possible neurotransmitter role for dopamine within the rat retina, and its possible interaction with glycine and taurine.

     

Trapped blood elements within the decidua of the rat pregnant uterus generate a lipoxygenase product(s) which inhibits myometrial prostacyclin synthesis

1 Prostacyclin (PGI₂) production by chopped segments of rat pregnant uterus was low compared with synthesis by separated myometrial tissue. Incubation of separated myometrium with decidua (2:1 by weight) led to an inhibition of myometrial PGI₂ output.

2 Boiling decidual tissue abolished the inhibitory influence on myometrial PGI₂ output. Preincubation of decidua with 5,8,11,14-eicosatetraynoic acid (ETA) (30 μg/ml) also suppressed decidual inhibitory activity but indomethacin (30 μg/ml) was ineffective.

3 Incubation of decidual and myometrial tissue with arachidonic acid (AA) 10 μg/ml did not increase the inhibition of myometrial PGI₂ synthesis, even if the decidua were pre-incubated with indomethacin.

4 Myometrial PGI₂ production was reduced if the chopped tissue was pre-incubated with soya bean lipoxidase for 10 min at 4°C. This reduction was reversed if the lipoxidase was incubated with ETA (30 μg/ml) for 30 min at 37°C before addition to the myometrial tissue.

5 Perfusion of the uterus to remove blood elements removed the inhibitory action that the decidua exerted upon myometrial PGI₂ production. PGI₂ synthesis by separated decidual and whole uterine tissue was markedly elevated.

6 The addition of rat blood platelets (0.75 × 10⁹/ml) to incubations of perfused decidual tissue reduced PGI₂ output and restored the inhibitory action that the decidua exerted on myometrial PGI₂ synthesis.

7 It is concluded that a lipoxygenase enzyme contained in blood platelets trapped within the decidual vasculature produces a hydroperoxy acid which inhibits decidual PGI₂ production or myometrial PGI₂ synthesis when the tissues are incubated together. It is suggested that perfusion is a pre-requisite before study of PGI₂ synthesis in highly vascularised tissues.

8 The pathophysiological importance of such platelet lipoxygenase products is discussed.

     

Antithrombotic effect of Z4A5 on coronary thrombosis in a canine model of acute unstable angina

Background and Purpose

The glycoprotein IIb/IIIa receptor is the final common pathway of platelet aggregation, regardless of the agonist, and thus represents an ideal therapeutic target for blocking coronary thrombosis. In this study, the anti-platelet and antithrombotic actions of Z4A5, a new glycoprotein IIb/IIIa receptor inhibitor, were evaluated in a canine model of acute unstable angina.

Experimental Approach

Z4A5 was given i.v. as a bolus followed by 60 min of continuous infusion at doses of 30 μg·kg⁻¹ + 1 μg·kg⁻¹·min⁻¹, 30 μg·kg⁻¹ + 5 μg·kg⁻¹·min⁻¹ or 300 μg·kg⁻¹ + 5 μg·kg⁻¹·min⁻¹. Its antithrombotic effect was evaluated in a model of coronary thrombosis, the injured, stenosed left circumflex coronary artery, in which platelet-dependent cyclic flow reductions (CFRs) were induced by vascular compression and constriction to simulate clinical acute unstable angina. Platelet aggregation and coagulation parameters were determined in platelet-rich plasma and platelet poor plasma respectively.

Key Results

The Z4A5 infusion induced a dose-dependent reduction in CFR frequency, which returned to baseline levels after the termination of the infusion at low doses. At medium dose that inhibited most part of platelet aggregation, it increased tongue bleeding time marginally with no dramatic changes in haemodynamic and coagulation parameters. Furthermore, the inhibition of ADP-induced platelet aggregation and prolonged bleeding time observed during Z4A5 infusion reverted to baseline levels after the termination of the infusion.

Conclusions and Implications

Z4A5 is an effective antithrombotic agent for coronary artery thrombosis with a rapid-on and rapid-off pharmacological profile, and could be used as an alternative treatment of coronary artery ischaemic syndromes.     

Distribution and types of adrenoceptors in the guinea-pig ileum: the action of alpha- and beta-adrenoceptor blocking agents

1 Transmurally stimulated segments of the guinea-pig ileum have been used to analyse the different adrenoceptors in the terminal (0 to 3 cm) and the proximal (> 50 cm from the ileocaecal valve) ileum.

2 The prejunctional adrenoceptors (located on the final, cholinergic, motor nerve terminals) and postjunctional adrenoceptors (located on the smooth muscle membrane) have been characterized according to their sensitivity to α- and β-agonists and antagonists.

3 Phentolamine, phenoxybenzamine and yohimbine, in concentrations of 0.1 μM, transiently enhanced (up to 10%) the twitch response. At higher concentrations all the α- and β-antagonists studied depressed the neurogenic twitches and relaxed the smooth muscle.

4 The twitch-inhibitory effects of adrenoceptor agonists (noradrenaline, adrenaline and ephedrine) were not antagonized by phenoxybenzamine (0.1, 0.5 and 1 μM), carbidine (0.5, 1 and 5 μM) and propranolol (0.5, 1 and 5 μM); however, they were depressed by phentolamine (0.1, 0.5, 1.25 and 5 μM) and yohimbine (0.25, 0.5 and 5 μM).

5 The smooth muscle contractions induced by noradrenaline and adrenaline in the terminal ileum and by phenylephrine in both the terminal and proximal ileum were antagonized by phenoxybenzamine, carbidine and phentolamine but were not influenced by yohimbine and propranolol.

6 The smooth muscle relaxations of the proximal ileum induced by noradrenaline, adrenaline and ephedrine were inhibited by yohimbine, phentolamine, carbidine and phenoxybenzamine, and the isoprenaline-induced relaxation was antagonized by propranolol.

7 All the agonists studied, except phenylephrine, elicited relaxations of the acetylcholine-induced sustained contraction of both proximal and terminal ileum. The relaxation induced by isoprenaline was antagonized by propranolol, and the effects of noradrenaline and ephedrine by yohimbine.

8 It is concluded that in the guinea-pig ileum there are postsynaptic β-adrenoceptors and at least two types of α-adrenoceptors: α₁-excitatory postjunctional adrenoceptors activated by phenylephrine, noradrenaline and adrenaline and antagonized by phenoxybenzamine, carbidine and phentolamine; α₂-inhibitory prejunctional adrenoceptors activated by ephedrine, noradrenaline and adrenaline and inhibited by yohimbine and phentolamine. The inhibitory postjunctional α-adrenoceptors are more close to the α₂-adrenoceptors, since they were stimulated predominantly by ephedrine and noradrenaline and inhibited by yohimbine.

9 It has been shown that all α-adrenoceptor subtypes are to be found at every distance (0 to 70 cm) from the ileocaecal valve and that they can be activated in the resting or in the acetylcholine-contracted states.

     

Catecholamine transport in isolated lung parenchyma of pig

1 Lung parenchyma strips of the pig incubated at 37°C with [³H]-(-)-noradrenaline ([³H]-NA) or [³H]-(±)-isoprenaline ([³H]-Iso), accumulated radioactivity via saturable, high affinity uptake processes. Apparent saturation constants (K_m) for [³H]-NA and [³H]-Iso were 1.34 × 10^-6 M and 1.63 × 10^-6 M respectively, while apparent transport maxima (V_max) were 4.86 and 1.63 × 10^-9 mol min^-1 g^-1 respectively.

2 Cellular accumulation of radioactivity from radiolabelled catecholamines was greatly reduced by lowering the temperature to 7°C, pretreatment with ouabain (100 μM), phentolamine (15 μM) or phenoxybenzamine (80 μM). However, accumulation of radioactivity derived from (³H]-NA was inhibited selectively by cocaine (10 μM) and desipramine (1 μM), while normetanephrine (80 μM) and 3-O-methylisoprenaline (50 μM) caused much greater reductions in cellular radioactivity from [³H]-Iso than from (³H]-NA. Taken together with information from kinetic studies, the results indicate that these amines are transported by separate uptake processes.

3 Cocaine (50 μM) which selectively reduced [³H]-NA transport, had no significant effect on the sensitivity (EC₅₀) of isolated parenchyma lung strips of the pig to the contractile effects of cumulative concentrations of NA. The catechol-O-methyl transferase (COMT) inhibitor, U-0521 (60 μM), also failed to alter the potency of NA, while normetanephrine (80 μM) caused a 2 fold decrease in potency.

4 Phentolamine (15 μM), which reduced the cellular accumulation of radioactivity derived from [³H]-Iso by 64%, caused a small potentiation of Iso-induced relaxations of porcine lung strips. Normetanephrine (80 μM) and 3-O-methylisoprenaline (50 μM), which also depressed the accumulation of cellular radioactivity from [³H]-Iso by > 50%, caused rightward shifts in Iso concentration-effect curves as a result of β-adrenoceptor blockade. In sharp contrast, cortisol (80 μM) and U-0521 (60 μM), which caused smaller reductions in the cellular accumulation of radioactivity derived from [³H]-Iso, both caused an approximately 9 fold potentiation of responses to Iso in isolated lung strips.

5 The results indicate that the major sites of uptake and metabolism of NA in porcine parenchyma strip are remote from α-adrenoceptors mediating NA-induced contraction. Similarly, some major sites of uptake of Iso are remote from β-adrenoceptors mediating Iso-induced relaxation. However, β-adrenoceptors are apparently in close proximity to a compartment containing COMT activity.

     

Comparative studies on the effects of tolmesoxide (Rx71107), a tolmesoxide metabolite (Rx71112) and nifedipine in isolated blood vessels

1 The effect was studied of tolmesoxide (Rx71107), a tolmesoxide metabolite (Rx71112) and nifedipine on active tension in human isolated crural veins and in rat thoracic aorta. The effects of tolmesoxide and nifedipine on ²²Na and ⁴⁵Ca net influx in noradrenaline-induced contractions were investigated in rat thoracic aorta.

2 Tolmesoxide, Rx71112 and nifedipine caused a concentration-related inhibition of noradrenaline (NA)- and potassium (K⁺)-induced contractions in human veins. Nifedipine was by far the most potent drug in these respects. Tolmesoxide (4.7 μM—4700 μM) and Rx71112 (22 μM—220 μM) inhibited the NA-induced contraction more effectively than the K⁺-induced concentration. The reverse was true for nifedipine (0.0023-3 μM).

3 Nifedipine had a similar potency in inhibiting the NA-induced contraction in rat aorta and human veins, whereas the inhibitory effect of tolmesoxide was more pronounced in rat aorta than in human veins.

4 Tolmesoxide was a weak antagonist of the contractile effects of the cumulative addition of calcium on human veins. In concentrations up to 470 μM, tolmesoxide was completely devoid of inhibitory effects on ²²Na and ⁴⁵Ca net influx in rat aorta. Rx71112 (220 μM) had an inhibitory effect on ²²Na net influx but no significant effect on ⁴⁵Ca net influx.

5 Nifedipine was an effective antagonist of the contractile effects of cumulative addition of calcium in human veins and had a concentration-related inhibitory effect on both ²²Na and ⁴⁵Ca uptake in rat aorta.

6 The results indicate that tolmesoxide and Rx71112 cause dilatation of human crural veins in vitro at concentrations that do not interfere with the net transmembranal movements of Ca and are thus clearly different from the effects of nifedipine.

     

Spectrum of the μ-, δ- and κ-binding sites in homogenates of rat brain

1 In homogenates of rat brain, the binding characteristics of tritiated opiates and opioid peptides were examined and the relative capacities of μ-, δ- and κ-binding sites of the opiate receptor determined by saturation analysis.

2 In competition experiments, binding of the selective μ-ligand [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin at the μ-site was displaced by [D-Ala²,D-Leu⁵]enkephalin with rather low affinity (K_I = 12.6 nM) and more readily by the ketazocine-like compounds (-)-ethylketazocine (K_I = 3.1 nM) and (-)-bremazocine (K_I = 0.32 nM), which also displaced the binding of [³H]-[D-Ala²,D-Leu⁵]enkephalin from the δ-site. In contrast, the binding to the κ-site was easily displaced by ethylketazocine (1.0 nM) and bremazocine (0.37 nM) but not by the μ-ligand [D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin (K_I = 2000-3000 nM) or the δ-ligand [D-Ala²,D-Leu⁵]enkephalin (K_I > 20,000 nM).

3 The dissociation equilibrium constant (K_D) and the binding capacity (pmol/g) of the μ-binding site were determined with the selective μ-ligand [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin. For the δ-site, [³H]-[D-Ala²,D-Leu⁵]enkephalin was used in the presence of unlabelled [D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin in order to suppress cross-reactivity to the μ-binding site. For the estimation of κ-binding, [³H]-(±)-ethylketazocine or [³H]-(-)-bremazocine were used in the presence of unlabelled μ- and δ-ligands for the suppression of cross-reactivities to the μ- and δ-binding sites.

4 In rat brain the capacity of the μ-binding site was 7.3 pmol/g brain, that of the δ-binding site 6.7 pmol/g brain and that of the κ-binding site 2.0 pmol/g brain. Thus, the κ-binding site had the lowest value whereas in the guinea-pig brain the capacity of the μ-binding site was lower than that of the δ- or κ-binding site.

     

Effects of the histamine H2-receptor blocking drugs burimamide and cimetidine on noradrenergic transmission in the isolated aorta of the rabbit and atria of the guinea-pig

1 In rabbit aortic strips, concentration-response curves to noradrenaline (NA) were shifted to the right in a parallel and concentration-dependent manner by the α-adrenoceptor blocking drug, phentolamine and also by the histamine H₂-receptor blocking drugs, burimamide and cimetidine. Responses to 5-hydroxytryptamine were not affected by these drugs.

2 Burimamide had the properties of a competitive antagonist of noradrenaline, possessing about one-hundredth the potency of phentolamine. Cimetidine was weaker than burimamide and did not fulfil the requirements for competitive antagonism of noradrenaline.

3 In guinea-pig isolated atria, in which noradrenergic transmitter stores were labelled with [³H]-noradrenaline, phentolamine (3 μM), burimamide (30 μM) and cimetidine (30 μM), in decreasing order of effectiveness, each enhanced stimulation-induced efflux of [³H]-noradrenaline, indicating that their blocking effects on prejunctional α-adrenoceptors in this tissue are in the same order of relative potency as on postjunctional α-adrenoceptors in rabbit aortic strips.

4 In the concentrations used (30 μM), neither burimamide nor cimetidine interfered with the neuronal uptake of noradrenaline. Burimamide, and to a much lesser extent, cimetidine, increased the resting efflux of [³H]-noradrenaline from guinea-pig atria.

5 The effect of clonidine, a partial agonist on prejunctional α-adrenoceptors in guinea-pig atria, in increasing stimulation-induced efflux of [³H]-noradrenaline when stimulated with 150 pulses at 5 Hz was blocked by cimetidine (30 μM) and reversed by phentolamine (3 μM) and burimamide (30 μM).

     

Inhibition of prostaglandin E1-responsive platelet adenylate cyclase by heparin: a study of the mechanism of inhibition and its relevance to platelet aggregation

1 Heparin can produce platelet aggregation in vitro and in vivo; it has been proposed that this may be due to the reported inhibition of the prostaglandin E₁ (PGE₁)-stimulated adenylate cyclase of the platelet by heparin.

2 The effect of heparin on the cyclic adenosine 3′,5′-monophosphate (cyclic AMP) response to PGE₁ was measured in intact and broken platelets both in vitro and in platelets obtained from normal subjects during intravenous infusion with herapin.

3 In platelet lysates, heparin produced a dose-related inhibition of PGE₁-stimulated adenylate cyclase. The maximum response to PGE₁ was reduced, with half-maximal inhibition occurring at 3 μg/ml heparin. This inhibition could be prevented by protamine sulphate.

4 Heparin did not affect PGE₁-stimulated cyclic AMP production in intact platelets either in vitro or in platelets taken during the infusion of 5,000iu heparin over 2h to 2 normal volunteers. Similarly, preincubation of platelets with heparin for up to 3h at 37°C did not affect platelet adenylate cyclase.

5 The effects of heparin were very similar to those of fluoride on the platelet adenylate cyclase: heparin and fluoride increased basal enzyme activity slightly (3-4 fold) but their effects were not additive; both inhibited the response to PGE₁ by approximately 50% when added directly to the assay and the inhibitory effects of the two were not additive; preincubation of membranes with either heparin or fluoride produced an irreversible state of inhibition.

6 As heparin inhibits PGE₁-stimulated adenylate cyclase activity only in broken platelets, we suggest that the aggregatory effects of heparin are probably independent of any action on cyclic AMP production.

     

Effects of sulphasalazine and its metabolites on prostaglandin synthesis, inactivation and actions on smooth muscle

1 We have investigated the effects of sulphasalazine and of its principal colonic metabolites (5-aminosalicylic acid and sulphapyridine) on prostaglandin inactivation, synthesis and actions on gastrointestinal smooth muscle.

2 Sulphasalazine inhibits prostaglandin F_2α breakdown in 100,000 g supernatants in all organs so far tested from 7 species with an ID₅₀ of approx. 50 μM; it has a selective action on prostaglandin 15-hydroxydehydrogenase and does not inhibit prostaglandin Δ-13 reductase, prostaglandin 9-hydroxydehydrogenase or `enzyme X' at millimolar concentrations. Enzyme activities were measured radiochemically or by bioassay.

3 Sulphapyridine and 5-aminosalicylic acid do not inhibit prostaglandin inactivation in vitro (4 species tested). A methyl analogue of sulphasalazine is a more potent inhibitor than the parent compound. Rabbit colon prostaglandin F_2α metabolism in vitro was inhibited by the following drugs with ID₅₀ values (μM) of: diphloretin phosphate 20, sulphasalazine 50, indomethacin 220, frusemide 1000 and aspirin 10,000. A similar rank order of potencies was obtained with rabbit kidney.

4 Sulphasalazine at 50 to 100 μM inhibited inactivation of prostaglandin E₂ in the perfused rat and guinea-pig lung by 3 to 40% (rat) and 32 to 100% (guinea-pig) when measured by superfusion cascade bioassay and of prostaglandin F_2α by 43.6 ± 6.5% in rat lung perfused with 50 μM sulphasalazine and assayed radiochemically.

5 Prostaglandins E₁ and E₂ were 97.0 ± 8.2% and 92.3 ± 6.8% inactivated in the lungs after intravenous injection in the anaesthetized rat as measured by reference to their vasodepressor potencies when injected intra-arterially. Prostaglandin A₂ was not similarly inactivated. Pulmonary inactivation was prevented in the presence of an intravenous infusion of 16.3 μg kg^-1 min^-1 sulphasalazine and partially inhibited at a lower infusion rate.

6 Prostaglandin biosynthesis from arachidonic acid was measured in microsomal preparations from four sources by bioassay and radiochemical methods. Indomethacin was a potent inhibitor (ID₅₀ 0.8 to 4.1 μM) but sulphasalazine and its methyl analogue were very weak inhibitors (ID₅₀ 1500 to > 5000 μM), 5-aminosalicylic acid was weaker still and sulphapyridine inactive.

7 Sulphasalazine at 50 μM did not affect the actions of prostaglandins on five smooth muscle preparations; at 500 μM there was a rapidly reversible and probably non-specific antagonism of responses to low doses of prostaglandins.

8 The specificity and selectivity of the interaction of sulphasalazine and its metabolites with the formation, breakdown and actions of prostaglandins are discussed.

     

Factors affecting the cerebrovascular response to noradrenaline in the dog

1 Noradrenaline infused into the internal carotid artery of the dog (0.01-1 μg kg^-1 min^-1) constricts the blood vessels of the cortex. This constriction is mediated by the action of noradrenaline on α-adrenoceptors of the cerebral arteries.

2 Intravenous (1 μg kg^-1 min^-1) or intra common carotid arterial (0.01-1 μg kg^-1 min^-1) infusions of noradrenaline cause an increase in cortical blood flow that can be dissociated from changes in blood pressure.

3 The effect of intravenous noradrenaline on the cortical blood vessels and metabolism is blocked by high PaCO₂ levels, or by the prior administration of (±)-propranolol. (+)-Propranolol is without such effect.

4 Following section of both vagi and both sinus nerves, intravenous noradrenaline fails to cause an increase in cortical blood flow.

5 In another series of animals the area of the carotid bifurcation was vascularly isolated and perfused with blood from a second dog. Chemoreceptor and baroreceptor activity was shown to be intact.

6 Administration of 5% CO₂ to the donor dog caused an increase in cerebral blood flow in the recipient dog.

7 Administration of intravenous noradrenaline (1.0 μg kg^-1 min^-1) to the donor animal caused an increase in cerebral blood flow, cerebral O₂ and glucose utilization of the recipient.

8 Administration of 5% CO₂ and intravenous (-)-noradrenaline (1.0 μg kg^-1 min^-1) caused a further increase in flow and metabolism.

9 This evidence suggests that the cerebrovasodilatation observed following intravenous noradrenaline is reflex and is triggered by chemoreceptor activity.

10 The evidence also suggests that the antagonism of the cortical dilatory effects of intravenous noradrenaline by raised PaCO₂ in the intact animal must be at a site different from the peripheral chemoreceptors.

     

Indirect action of 5-hydroxytryptamine on the isolated muscularis mucosae of the guinea-pig oesophagus

1 The site of action of 5-hydroxytryptamine (5-HT) was examined on the isolated muscularis mucosae attached to the submucous plexus of the guinea-pig oesophagus. Isotonic responses of the longitudinal muscularis mucosae were recorded.

2 5-HT produced a transient contraction of the muscularis mucosae at concentrations higher than 3 μM. The contraction was rapid in onset, reaching a peak in about 15 s or less, and was restored to the basal level after 20 to 30 s without washing out 5-HT. When the 5-HT-induced contraction faded to the basal tone, successive applications of 5-HT no longer produced any contracture.

3 Nicotine (Nic), at concentrations higher than 10 μM, also produced a transient contraction which had a very similar pattern to that induced by 5-HT. Again, the successive application of Nic no longer produced any contracture following prior treatment with Nic itself. However, the 5-HT-induced contraction was not modified by the presence of Nic.

4 Exogenously applied acetylcholine (ACh) produced a concentration-dependent contraction of the muscularis mucoase, the 50% effective concentration (EC₅₀) was 69 ± 5.6 nM. The contraction was sustained during incubation with ACh, and was not modified by prior treatment with 5-HT or Nic.

5 The 5-HT (100 μM)-induced contraction was completely abolished by tetrodotoxin (0.2 μM) and atropine (0.2 μM). This means that the action is mediated by stimulating cholinergic nerves in the submucous plexus attached to muscularis mucosae. Moreover, the stimulating action of 5-HT does not involve nicotinic receptors, since the action was not blocked by hexamethonium (100 μM).

6 Among several 5-MT antagonists examined, methysergide (1 μM), ketanserin (1 μM) and morphine (100 μM) failed to modify the 5-HT (100 μM)-induced contraction significantly. Cinanserin (0.1-3 μM), cyproheptadine (3-100 nM) and phenoxybenzamine (0.1-3 μM) inhibited the 5-HT-induced contraction, in a concentration-dependent manner, and each highest concentration abolished the response. However, none of these antagonists was specific for 5-HT, but the Nic (100 μM) or ACh (0.1 μM)-induced contractions were also inhibited by them.

7 The present results indicate that 5-HT contracts the muscularis mucosae of the guinea-pig oesophagus indirectly by stimulating cholinergic nerves in the submucous plexus, and has no direct action on the muscularis mucosae. In addition, the type of 5-HT receptors responsible for the stimulant action may be different from those in other parts of the gastrointestinal tract, blood vessels or brain, because of the different effects of 5-HT antagonists.

     

Anti-thrombotic effects and bleeding risk of AJvW-2, a monoclonal antibody against human von Willebrand factor

1. A murine anti-human vWF monoclonal antibody, AJvW-2, was developed that inhibited the interaction between platelet glycoprotein Ib (GPIb) and von Willebrand factor (vWF) during the ristocetin- (IC₅₀=0.7±0.1 μg ml⁻¹) and botrocetin- (IC₅₀=1.8±0.3 μg ml⁻¹) induced aggregation of human platelets.
2. AJvW-2 inhibited the high shear stress (10.8 N m⁻²) induced aggregation of human platelets dose-dependently with an IC₅₀=2.4±0.3 μg ml⁻¹, but had no effect on low shear stress induced platelet aggregation (1.2 N m⁻²) up to 100 μg ml⁻¹.
3. AJvW-2 also inhibited the high shear stress (5.0 N m⁻²) induced adhesion of human platelets to collagen I with the same efficacy (IC₅₀=2.4±0.3 μg ml⁻¹), but had no effect at low shear conditions (1.5 N m⁻²).
4. AJvW-2 inhibited the botrocetin-induced aggregation of platelets from guinea-pig, rat, rabbit, dog and pig at the same concentration range as human platelets; it likewise also inhibited the high shear stress induced aggregation and adhesion to collagen I of guinea-pig platelets.
5. AJvW-2 prevented arterial thrombus formation in guinea-pigs at a dose of 100 μg kg⁻¹ without prolonging the template bleeding time, whereas the GPIIb/IIIa antagonist lamifiban mediated inhibition of thrombosis at 1000 μg kg⁻¹ was accompanied by a significant prolongation of the bleeding time.
6. These results suggest that AJvW-2 is a potent inhibitor of the GPIb-vWF interaction and a potential novel antithrombotic agent with lower bleeding risk than GPIIb/IIIa antagonists.

     

Effects of adenosine analogs and adenine nucleotides on adenosine 5'-diphosphate-induced rat platelet aggregation

Various adenosine analogs and adenine nucleotides have been tested as inhibitors of ADP-induced aggregation of rat platelets. The potent inhibitors of human platelet aggregation, adenosine, 2-fluoroadenosine, 2-chloroadenosine, carbocyclic adenosine and N⁶-phenyl adenosine, had little effect on rat platelet aggregation (0–30 per cent inhibition). The effects of adenosine or its analogs on ADP-induced aggregation of cross-species platelet-rich plasmas (PRPs) (human platelets suspended in rat plasma or rat platelets in human plasma) were similar to those with the native PRPs, indicating that these species differences were due to intrinsic factors in the platelets and not in the plasma. When these analogs were tested in the presence of the cyclic AMP phosphodiesterase inhibitor papaverine, strong inhibiton of rat platelet ADP-induced aggregation was seen. 2′-Deoxyadenosine and 3′-deoxyadenosine were not inhibitory to ADP-induced aggregation of rat PRP even in the presence of papaverine. Adenosine 5′-tetraphosphate strongly inhibited both human and rat platelet aggregation. AMP, like adenosine, did not inhibit rat platelet aggregation but became strongly inhibitory in the presence of papaverine. This inhibitory effect was abolished by preincubating rat PRP with an adenylate cyclase inhibitor, 2′, 5′-dideoxyadenosine or adenosine deaminase. In the later case, however, if the adenosine deaminase inhibitor 2′-deoxycoformycin was included in the incubation mixture, the inhibition by AMP plus papaverine was similar to adenosine plus papaverine. About 50 per cent of [¹⁴C]AMP was converted to [¹⁴ C]adenosine in rat platelet-free plasma or PRP after a 10-min incubation. α,β-Methylene-ADP and β,γ-methylene-ATP (200 μM) inhibited rat platelet aggregation by 50 and 64 per cent, respectively. Cyclic AMP phosphodiesterase of rat and human platelets gave comparable K_m, and V_max values (K_m 0.53 and 0.21μM and V_max 6.0 and 6.7 pmoles/min/10⁷ platelets, respectively).     

The effects of three β-adrenoceptor blocking drugs on isolated preparations of skeletal and cardiac muscle

1 The effects of propranolol, oxprenolol and practolol on the isometric twitch responses to electrical stimulation of isolated diaphragm muscles from the rat and of isolated papillary muscles from the rabbit are described.

2 Depression of the twitch responses of the diaphragm muscle was produced by propranolol (20 μg/ml), by oxprenolol (100 μg/ml) and by practolol (500 μg/ml).

3 Depression of the twitch responses of the papillary muscles was produced by propranolol (20 μg/ml) by oxprenolol (100 μg/ml) and by practolol (200 μg/ml).

4 No increase of twitch tension was produced by oxprenolol or practolol on either tissue.

5 It is concluded that propranolol, oxprenolol and practolol produce negative inotropic actions on isolated cardiac muscle by a mechanism unrelated to blockade of β-adrenoceptors and which occurs at doses which are well in excess of those doses required to produce β-blockade.

     

The haemoglobin-oxygen dissociation curve: In vivo and in vitro effects of five β-adrenoceptor antagonists and lignocaine

1 The effects of several β-adrenoceptor antagonists and lignocaine on blood oxyhaemoglobin dissociation curves have been studied in healthy non-smoking subjects.

2 The P_O2 at 50% saturation (P₅₀) did not significantly change after oral propranolol 80 mg (single dose, and following 2 weeks' administration of 80 mg twice daily), or following separate intravenous injection of propranolol (0.2 mg/kg), practolol (1 mg/kg), atenolol (0.2 mg/kg) and SL 75212 (0.15 and 0.6 mg/kg).

3 Increases in P₅₀ were found after the addition of propranolol 100 and 500 μg/ml, and lignocaine 5 μg/ml to whole blood, but incubation with propranolol at 1000 ng/ml or less, or sotalol 5 and 500 μg/ml and 5 mg/ml resulted in no significant change in P₅₀.

4 These results suggest that to increase P₅₀ with propranolol requires plasma propranolol concentrations far in excess of concentrations normally achieved, and that the therapeutic effect of propranolol in patients with ischaemic heart disease cannot be attributed to an increase in P₅₀.

     

In vitro adrenaline and collagen-induced mobilization of platelet calcium and its inhibition by naftopidil, doxazosin and nifedipine

Aims The aim of the study was to obtain further information regarding the modes of action of doxazosin, naftopidil and nifedipine on platelet function.Methods We conducted an in vitro study of drug influences on adrenaline and collagen-induced mobilization of platelet calcium.‘fn2\Results In the presence of fibrinogen (300 μg ml⁻¹ ) both collagen (5 μg ml⁻¹ ) and adrenaline (16 μm ) stimulated the aggregation of washed platelets. Collagen induced a transient rise (+4.97±0.63 μm ) in platelet Ca²⁺ concentration, [Ca²⁺]_i, as measured using the photoprotein aequorin, which coincided with the onset of aggregation. Adrenaline induced a smaller rise (+3.6±0.96 μm ) which, however, occurred after the onset of aggregation. Naftopidil, an α₁-adrenoreceptor antagonist produced a concentration-dependent inhibition of collagen-induced Ca²⁺ mobilization, maximum inhibition (22.9±4%, P<0.05) occurring with 40 μm naftopidil. The inhibition of Ca²⁺ mobilization was not reflected by a concentration-dependent inhibition of platelet aggregation, although 40 μm naftopidil produced statistically significant inhibition (23.3±11.7%, P<0.05). The adrenaline-induced rise in [Ca²⁺]_i was inhibited dose dependently by naftopidil (e.g. 40 μm naftopidil, 100±0%, P<0.05), as was aggregation (40 μm naftopidil, 100±0%, P<0.05). Doxazosin, another α₁-adrenoreceptor blocker, inhibited Ca²⁺ mobilization induced by collagen to similar extents as for naftopidil (30 μm doxazosin, 17.4±2.5%, P<0.05), but did not inhibit platelet aggregation. It also inhibited the adrenaline-induced rise in [Ca²⁺]_i in a concentration-dependent manner (30 μm doxazosin, 37.6±13.7%, P<0.05), significant inhibitions of platelet aggregation also being produced (30 μm, 49.6±17.2%, P<0.05). As expected, the calcium channel blocker nifedipine produced concentration-dependent inhibitions of both collagen-induced Ca²⁺ mobilization (e.g. 28 μm nifedipine, 47.8±2.7%, P<0.05) and aggregation (28 μm, 55.1±9.2%, P<0.05).Conclusions These data indicate that the α₁-adrenoreceptor blockers, naftopidil and doxazosin, inhibit Ca²⁺ mobilization, this mechanism being possibly the means whereby these drugs inhibit platelet aggregation.