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1 In the present study we have classi®ed the receptor(s) mediating increases in intracellular calcium concentration ([Ca 2+ ] i ) in human washed platelets and compared the pharmacological pro®le obtained with that observed in Jurkat cells, stably transfected with a bovine P2Y 1 -receptor. 2 The P2Y 1 -receptor antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P), competitively antagonized agonist responses in both Jurkat cells, and in platelets with similar a nities (pK B of 5.8 and 6.0, respectively). 3 The selective P2Y ADP antagonist, AR-C66096, exhibited partial agonism in the Jurkat cells with an a nity (pK A ) of 4.9. This value is consistent with its known P2Y 1 -receptor activity. In platelets, AR-C66096 at a concentration (0.1 mM) approximately 100 fold greater than its known P2Y ADP receptor a nity, had no e ect on ADP-induced increases in [Ca 2+ ] i . 4 The ability of adenine nucleotide analogues to elevate [Ca 2+ ] i in the Jurkat cells was also determined. The rank order of agonist potency (p[A] 50 ) was: 2-MeSADP (8.3)42-ClATP (7.8)4ADP (7.5)=2-MeSATP (7.4)4ATPgS (6.5)4ATP (6.2), with ATP appearing to be a partial agonist. 5 The same rank order of potency was observed when similar experiments were performed in platelets. However, the absolute potencies of all the agonists and the intrinsic activities of both ATPgS and ATP were lower in platelets. 6 The operational model of agonism was used to test whether the agonist concentration-e ect pro®les obtained in these two cell types could be explained on the basis of di erences in receptor reserve. The analysis indicated that the data obtained in platelets closely resembled that predicted for a low density or poorly coupled P2Y 1 -receptor system. 7 The hypothesis that the observed partial agonist behaviour of ATP was the result of receptor activation by contaminating ADP with concomitant receptor blockade by ATP, was tested in the platelet system. This hypothesis was supported by a theoretical analysis, which yielded an a nity value for ATP similar to that obtained previously at P2Y 1 -receptors. 8 In summary, the results of this study indicate that human washed platelets contain P2Y 1 -receptors which mediate increases in [Ca 2+ ] i and that this receptor population is pharmacologically distinct from P2Y ADP -receptors.

In normal physiologic responses to injury and infection, inflammatory cells enter tissue and sites of inflammation through a chemotactic process regulated by several families of proteins, including inflammatory chemokines, a family of small inducible cytokines. In neutrophils, chemokines chemokine (CXC motif) ligand 1 (CXCL1) and CXCL8 are potent chemoattractants and activate G protein-coupled receptors CXC chemokine receptor 1 (CXCR1) and CXCR2. Several small-molecule antagonists of CXCR2 have been developed to inhibit the inflammatory responses mediated by this receptor. Here, we present the data describing the pharmacology of 3S)-3,4-dihydroxybutan-2-yloxy)pyrimidin-4-yl)azetidine-1-sulfonamide], a novel antagonist of CXCR2. AZD5069 was shown to inhibit binding of radiolabeled CXCL8 to human CXCR2 with a pIC 50 value of 9.1. Furthermore, AZD5069 inhibited neutrophil chemotaxis, with a pA 2 of approximately 9.6, and adhesion molecule expression, with a pA 2 of 6.9, in response to CXCL1. AZD5069 was a slowly reversible antagonist of CXCR2 with effects of time and temperature evident on the pharmacology and binding kinetics. With short incubation times, AZD5069 appeared to have an antagonist profile with insurmountable antagonism of calcium response curves. This behavior was also observed in vivo in an acute lipopolysaccharide-induced lung inflammation model. Altogether, the data presented here show that AZD5069 represents a novel, potent, and selective CXCR2 antagonist with potential as a therapeutic agent in inflammatory conditions.

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1 The effects of initial stretch and degree of agonist-induced tone on acetylcholine-induced relaxations were examined in rings of rat isolated aorta. The relaxation to acetylcholine was antagonized by atropine and almost completely abolished by haemoglobin. Relaxation to sodium nitroprusside was similar in rings with an intact or disrupted endothelium but that to isoprenaline was greater in intact preparations. 2 In preparations with either an intact or disrupted endothelium there was a similar length-dependent increase in the resting tension of the aortic rings. The size of the contractile response to phenylephrine (1 pM) was dependent on the initial length (and hence degree of stretch) of the preparation in both rubbed and unrubbed tissues. The absolute difference in contractile response between rubbed and unrubbed was greatest at 1.8mm and less at the other lengths tested, including the optimum degree of stretch for contraction i.e. 2.4 mm. 3 The absolute acetylcholine-induced relaxation (only seen in rings with an intact endothelium) was dependent on the initial length (and hence degree of stretch) of the preparation and was maximum at 2.4 mm. The proportionate relaxation (i.e. expressed as a percentage of induced tone) was also lengthdependent, being optimal at 1.5 mm. 4 The sensitivity of the vessels to acetylcholine varied depending on the level of agonist-induced tone.When tone was low, acetylcholine sensitivity was high (at [NA] 0.03 pM: pIC50 = 7.36 + 0.07), when the concentration of noradrenaline was increased the tone increased and the acetylcholine sensitivity was low (at [NA] 0.3jum: p'C50 = 6.57 + 0.07). 5The absolute sensitivities and maximum relaxations induced by acetylcholine are discussed in relation to the initial degree of stretch (and hence length of the preparation) or the degree of agonist-induced tone.

2+ ] i elevation but the e ects of UTP (E max of IL-8 response increased to 50+1% of the maximal response to ATP, pEC 50 increased to 9.8+0.1) were greater than those of 2MeSADP (E max increased to 36+2%, pEC 50 increased to 8.7+0.2). 5 The potentiation of IL-8-mediated Ca 2+ signalling by UTP was not dependent upon the time of IL-8 addition following UTP but was dependent on the continued presence of UTP. Potentiated IL-8 Ca 2+ signalling was apparent in the absence of extracellular Ca 2+ , demonstrating the release of Ca 2+ from intracellular stores. 6 Activation of P2Y1 and P2Y2 receptors also revealed Ca 2+ signalling by an endogenously expressed, Ga s -coupled b-adrenoceptor. 7 In conclusion, pre-stimulation of P2Y nucleotide receptors, particularly P2Y2, facilitates Ca 2+ signalling by either recombinant CXCR2 or endogenous b-adrenoceptors.

Background and purpose: b2-Adrenoceptor agonists (b2-agonists) are important bronchodilators used in the treatment of asthma and chronic obstructive pulmonary disease. At the molecular level, b2-adrenergic agonist stimulation induces desensitization of the b2-adrenoceptor. In this study, we have examined the relationships between initial effect and subsequent reduction of responsiveness to restimulation for a panel of b2-agonists in cellular and in vitro tissue models. Experimental approach: b2-Adrenoceptor-induced responses and subsequent loss of receptor responsiveness were studied in primary human airway smooth muscle cells and bronchial epithelial cells by measuring cAMP production. Receptor responsiveness was compared at equi-effective concentrations, either after continuous incubation for 24 h or after a 1 h pulse exposure followed by a 23 h washout. Key findings were confirmed in guinea pig tracheal preparations in vitro. Key results: There were differences in the reduction of receptor responsiveness in human airway cells and in vitro guinea pig trachea by a panel of b2-agonists. When restimulation occurred immediately after continuous incubation, loss of responsiveness correlated with initial effect for all agonists. After the 1 h pulse exposure, differences between agonists emerged, for example isoprenaline and formoterol induced the least reduction of responsiveness. High lipophilicity was, to some extent, predictive of loss of responsiveness, but other factors appeared to be involved in determining the relationships between effect and subsequent loss of responsiveness for individual agonists. Conclusions and implications: There were clear differences in the ability of different b2 agonists to induce loss of receptor responsiveness at equi-effective concentrations.

1 The action of methoctramine and himbacine at muscarinic receptors has been studied using guinea-pig isolated trachea, oesophageal muscularis mucosae, paced left atria, and rat aortic preparations.2 Methoctramine (1 x 10-6-3.2 x 10-0M), but not himbacine, elicited positive inotropic responses. These responses were enhanced by pretreating the animals with reserpine. The responses in reserpine-treated animals were not antagonized by phentolamine (1 x 10-6M) but were antagonized by propranolol (1 x 10-6M).3 Methoctramine, but not himbacine, exhibited allosteric inhibitory effects at cardiac muscarinic receptors, resulting in a curvilinear Schild plot. Deviations from competitive antagonism were also observed in combination dose-ratio experiments using atropine and methoctramine. At 1 x 10-6M, the pKB value for methoctramine was 7.88 + 0.15 (mean + s.e.mean, n = 5). The pA2 value for himbacine at cardiac muscarinic receptors was 8.52 + 0.06 (n = 3). 4 At tracheal and oesophageal muscularis mucosal smooth muscle receptors, the Schild plots for both antagonists were linear. The pA2 values for methoctramine at receptors in these two preparations were similar (6.08 + 0.05 and 6.03 + 0.09 respectively, n = 4) and were approximately 60 fold less than those values observed at atrial receptors. Himbacine, also exhibited similar values at muscarinic receptors in the trachea and oesophageal muscularis mucosae (7.61 + 0.05 and 7.57 + 0.04 respectively, n = 4). 5 Muscarinic receptors mediating relaxation of the rat aortic endothelium exhibited pA2 values for methoctramine (5.87 + 0.12, n = 6) which were similar to those observed in the smooth muscle, but not the atria. The pA2 values for himbacine at endothelial muscarinic receptors were approximately 0.5 pA2 units lower than those observed at muscarinic receptors in smooth muscle (6.92 + 0.80, n = 6). In addition, the Schild slopes for methoctramine and himbacine at these receptors were significantly (P < 0.05) less than unity. 6 Methoctramine, and to a lesser extent himbacine, are potent and selective antagonists for cardiac muscarinic receptors. However, caution should be used in interpretation of the data with methoctramine in view of the inhibitory allosteric properties and direct inotropic actions of this compound.