1 BW A868C, a novel compound, behaved as a simple competitive antagonist in a human washed platelet aggregation assay. Anti-aggregatory concentration-effect curves to BW 245C were displaced in a parallel manner. The shifts accorded with a Schild plot slope of unity and a pKB of 9.26. 2 Inhibition of platelet aggregation by prostaglandin D2 (PGD2) was antagonized with a similar potency, as were the relaxation effects of BW 245C and PGD2 in the rabbit jugular vein. BW A868C can, therefore, be classified as a DP-receptor antagonist.3 Actions of BW A868C at other prostaglandin receptors (IP, EP,, EP2, TP and FP) were excluded at concentrations up to 1,000 times higher than the DP-receptor affinity. 4 Analyses of BW 245C-and PGD2-mediated effects were complicated by additional agonist receptor interactions which were revealed by BW A868C. In rabbit jugular vein a resistant phase of agonism was detectable, indicating that both agonists exerted effects through another receptor (possibly EP2). Also, PGD2, in addition to its anti-aggregatory effect on platelets, demonstrated a pro-aggregatory action in the presence of BW A868C. 5 The contractile effects of PGD2 in guinea-pig tracheal strips were resistant to 10 tM BW A868C indicating that they were not mediated through DP-receptors. 6 To our knowledge this is the first account of a well-classified competitive antagonist at the DP-receptor. Its potency and selectivity make it an important new tool in prostanoid receptor classification and identification.
1 Isolated rings of rabbit external jugular vein (RbJV) and rat thoracic aorta (RA) were used to study the effect of the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME) on muscarinic and 5-hydroxytryptamine (5-HT) receptor-stimulated, endothelium-dependent vascular relaxations. 2 In RbJV relaxations produced by the endothelial 5-HT receptor agonist a-methyl-5-HT were potently and non-surmountably inhibited by L-NAME (10pM), whereas acetylcholine relaxations in this tissue were unaffected by this concentration of inhibitor. By contrast, acetylcholine relaxations in RA were virtually abolished by 1O0 M L-NAME. In each case an equivalent concentration of D-NAME was without effect on agonist-induced relaxations. 3 The different effect of L-NAME on acetylcholine relaxations in RbJV and RA was not due to muscarinic receptor differences. Affinity estimates for acetylcholine (pKA = 6.12 + 0.09; 6.09 + 0.08 respectively) and for 4-diphenyl-acetoxy-N-methylpiperidine methobromide (4-DAMP, pKB = 9.01 + 0.12; 9.24 + 0.16 respectively) indicated that the receptors in both tissues belong to the same M3 class. Tissue differences resulting from the release of a cyclo-oxygenase product or a. glibenclamide-sensitive K+-channel-linked hyperpolarizing factor were also ruled out by selective inhibition of these pathways. 4 When phenoxybenzamine was used to reduce the efficacy of acetylcholine in RbJV so that it behaved as a partial agonist in this tissue, L-NAME (10pM) now produced non-surmountable inhibition of relaxation responses. In untreated tissues the same concentration of L-NAME also profoundly inhibited responses produced by butyrylcholine and pilocarpine, both of which behave as partial agonists at the M3 receptor in RbJV. 5 A simple model was developed which describes the theoretical behaviour of receptor-stimulated synthesis and release of NO. The model predicts that competitive inhibition of NO formation results in parallel displacements of the agonist response curve in the case of high efficacy agonist, but right-shift with concomitant depression of the curve maximum in the case of low efficacy agonists. Simulations based on the model showed reasonable agreement with the experimental data. 6 It is concluded that analogues of L-arginine demonstrate tissue-and agonist-dependence in terms of their ability to inhibit receptor-mediated events involving the liberation of NO. This behaviour can reflect differences in agonist efficacy in the receptor systems being studied, a possibility that should be ruled out before apparent resistance to inhibition is taken as evidence for the involvement of heterogeneous endothelium-derived relaxing factors (EDRFs).
1 Chinese hamster ovary (CHO) cells have been reported to be devoid of 5-HT receptors and have frequently been used as hosts for the expression of cloned 5-HT receptors. Unexpectedly, 5-HT was found to induce profound inhibition of forskolin-stimulated cyclic AMP production in these cells and the aim of this study was to classify the 5-HT receptor involved. 2 In CHO(dhfr-) cells 5-HT was a potent agonist and caused 80-100% inhibition of forskolin stimulated cyclic AMP production. A study using several 5-HT, receptor
1 The aim of this study was to determine the receptor profile of 9all1i-prostaglandin F2 (PGF2) and compare it with that of its parent, prostaglandin D2 (PGD2 I-PGF2 and PGD2 (10pM) were without affinity at the IP receptors on human platelets and had no agonist action in the EP3 receptor containing preparation, guinea-pig vas deferens. 6 9ax1 1,-PGF2 is a major metabolite of PGD2 in vivo and this conversion clearly represents an inactivation step since 9aIllfl-PGF2 is of considerably lower potency than PGD2 at DP receptors. However, it is of similar potency to PGD2 at TP, FP, EP1 and EP2 receptors and it may, therefore, contribute to the biological effects which follow PGD2 administration or endogenous synthesis; its actions at these receptors are likely to be similar to those of PGD2 itself.
The ability of 5-hydroxytryptamine (5-HT) to augment contractions evoked by other agents (and vice versa) in vascular smooth muscle has been studied for many years (see [ 1, 21 for reviews). The abundance of the data available contrasts starkly with the lack of knowledge of the mechanisms underlying such amplification phenomena. Our own interest in 5-HT stems from not only the proposed involvement of this amine in cardiovascular disease involving platelet aggregation, but also from the more recent knowledge that both 5-HT, and 5-HT, receptors mediate contraction of vascular smooth muscle. Thus, there is a multitude of possible interactions between the various factors released from aggregating platelets. We have examined the interactions between the plateletderived products 5-HT and thromboxane A, (TxA,), which are found in elevated concentrations in the plasma of patients with cardiovascular disease [3, 41. Interactions between 5-HT and TxA, were studied in the rabbit femoral artery [5, 61. Briefly, vascular ring segments were suspended in organ baths filled with Krebs' saline for recording isometric force changes. Abbreviations used: A, agonist; a-Me-5-HT, ( + ) -amethyl-5-hydroxytryptamine; 5-HT, 5-hydroxytryptamine; NPY, neuropeptide Y; p[Aji,j3 midpoint location of agonist concentration-effect curves; PIP2, phosphatidylinositol 4,s-bisphosphate; TxA2, thromboxane A?. 'To whom correspondence should be addressed. benextramine tetrahydrochloride (3 p M for 30 min). Interactions were examined by inducing contraction corresponding to lo%, 30% or 50% of the tissue maximum with one agonist (A,,, A,, and /I5,) and, at steady state, a concentration-effect curve constructed to a second agonist. Interactions between 5-HT, and TxA, receptor-mediated responsesConcentration-effect curves to the selective 5-HT, receptor agonist f a-methyl-5-hydroxytryptamine (a-Me-5-HT) were obtained in the presence and in the absence of the stable TxA,-mimetic U46619 (Figure 1 a). Responses to a-Me-5-HT {control midpoint location (p[A,,,]) = 6.82 -t 0.04) were displaced significantly to the left in the presence of U46619, the maximum change in midpoint location occurred when the tissues were contracted with U46619 to 30% of their maximum (p[A,,,] = 7.24 f 0.09). At the highest concentration of U466 19 (AS,) the a-Me-5-HT midpoint location moved back towards that of the control (p[A5,] = 7.16 f 0.07).There was no significant increase in the maximum response to a-Me-5-HT at any concentration of U46619. These data can be illustrated further in the form of histograms showing the effects of different concentrations of a-Me-5-HT and U466 19, alone and in combination (Figure lb). This shows that, at lower concentrations only, the effect of these two agents when applied in combination is greater than the simple sum of their individual effects.The interaction between agonists acting at 5-HT, and at TxA, receptors are consistent with the expectations of threshold synergy [5]. A theoretical model of threshold synergy [7] proposes that if the trans...
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