Kenneth L. Clark

EP4 prostanoid receptor-mediated vasodilatation of human middle cerebral arteries

Dilatation of the cerebral vasculature is recognised to be involved in the pathophysiology of migraine. Furthermore, elevated levels of prostaglandin E2 (PGE2) occur in the blood, plasma and saliva of migraineurs during an attack, suggestive of a contributory role. In the present study, we have characterised the prostanoid receptors involved in the relaxation and contraction of human middle cerebral arteries in vitro. In the presence of indomethacin (3 μM) and the TP receptor antagonist GR32191 (1 μM), PGE2 was found to relax phenylephrine precontracted cerebral arterial rings in a concentration‐dependent manner (mean pEC50 8.0±0.1, n=5). Establishment of a rank order of potency using the EP4>EP2 agonist 11‐deoxy PGE1, and the EP2>EP4 agonist PGE1‐OH (mean pEC50 of 7.6±0.1 (n=6) and 6.4±0.1 (n=4), respectively), suggested the presence of functional EP4 receptors. Furthermore, the selective EP2 receptor agonist butaprost at concentrations <1 μM failed to relax the tissues. Blockade of EP4 receptors with the EP4 receptor antagonists AH23848 and EP4A caused significant rightward displacements in PGE2 concentration–response curves, exhibiting pA2 and pKB values of 5.7±0.1, n=3, and 8.4, n=3, respectively. The IP receptor agonists iloprost and cicaprost relaxed phenylephrine precontracted cerebral arterial rings (mean pEC50 values 8.3±0.1 (n=4) and 8.1±0.1 (n=9), respectively). In contrast, the DP and FP receptor agonists PGD2 and PGF2α failed to cause appreciable relaxation or contraction at concentrations of up to 30 μM. In the absence of phenylephrine contraction and GR32191, the TP receptor agonist U46619 caused concentration‐dependent contraction of cerebral artery (mean pEC50 7.4±0.3, n=3). These data demonstrate the presence of prostanoid EP4 receptors mediating PGE2 vasodilatation of human middle cerebral artery. IP receptors mediating relaxation and TP receptors mediating contraction were also functionally demonstrated.

Pharmacological profile of GR117289 in vitro: a novel, potent and specific non‐peptide angiotensin AT1 receptor antagonist

1 This paper describes the effects of GR117289 (1‐[[3‐bromo‐2‐[2‐(1H‐tetrazol‐5‐yl)phenyl]‐5‐benzofuranyl]methyl]‐2‐butyl‐4‐chloro‐1H‐imidazole‐5‐carboxylic acid) at angiotensin receptors and binding sites in rabbit aorta, rat liver and bovine cerebellum preparations in vitro. 2 In rabbit isolated aortic strips, GR117289 (0.3, 1 and 3 nm) caused a concentration‐related, insurmountable suppression of the concentration‐response curve to angiotensin II (AII). When the contact time was increased, a greater degree of antagonism of AII was observed, suggesting that GR117289 is slow to reach equilibrium. A pKB of 9.8 ± 0.1 was calculated for GR117289 after 3 h incubation. GR117289 (1 μm) did not affect contractile responses to phenylephrine or 5‐hydroxytryptamine (5‐HT) in the rabbit aorta. 3 GR117289 (1 nm) alone caused a marked suppression and a slight rightward displacement of the AII concentration‐response curve. Co‐incubation with the competitive, surmountable AT1 receptor antagonist, losartan (10 nm, 100 nm and 1 μm), resulted in a concentration‐related upward and rightward displacement of the concentration‐response curve to subsequently administered AII. In separate experiments in which preparations were pre‐incubated with GR117289 (1 nm), subsequent addition of losartan (1 μm) for 2, 15 or 45 min caused a further, but similar, rightward displacement of the concentration‐response curve to subsequently administered AII with a time‐dependent increase in the maximum response. 4 Suppression of AII‐induced contractile responses, caused by superfusion with GR117289 (0.3, 1 or 3 nm) was not reversed by continuously washing the tissues for 3 h; in fact, the potency of GR117289 was slightly enhanced after this period. 5 In rat liver membranes, GR117289 was a potent competitor with [3H]‐AII for AT1 binding sites (pKi = 8.7 ± 0.1) but in bovine cerebellum membranes, it was a very weak competitor for AT2 binding sites (pKi < 6). Pre‐incubation of rat liver membranes with GR117289 had little effect on its affinity (pKi = 9.1 ± 0.21), but increasing the concentration of bovine serum albumen in the assay buffer from 0.001% to 0.1% w/v decreased affinity (pKi = 7.5 ± 0.1). 6 In saturation binding experiments in rat liver membranes, GR117289 (12 nm) increased the Kd of [3H]‐AII from 0.28 ± 0.06 nm to 0.37 ± 0.02 nm, and decreased Bmax from 10.0 ± 0.1 to 5.6 ± 0.3 fmol mg−1 tissue. In other experiments, GR117289 (1 μm) did not alter the rate of dissociation of [3H]‐AII from AT1 binding sites, following addition of excess unlabelled AII. 7 In rabbit aorta vascular smooth muscle membranes, GR117289 competed with [125I]‐Sar1Ile8 AII for binding to AT1 binding sites. In the presence of 0.1% w/v bovine serum albumen, a pIC50 of 7.6 ± 0.1 was calculated. Under the same conditions, but with rat liver membranes, a pIC50 of 7.8 ± 0.1 was determined. 8 Taken together, these results show that GR117289 is a potent, specific, selective and insurmountable antagonist at angiotensin AT1 receptors. Its profile in the rabbit aorta is consistent with the proposal that GR117289 is a slowly reversible (pseudo‐irreversible) antagonist at these receptors.

Evaluation of low density array technology for quantitative parallel measurement of multiple genes in human tissue

BackgroundLow density arrays (LDAs) have recently been introduced as a novel approach to gene expression profiling. Based on real time quantitative RT-PCR (QRT-PCR), these arrays enable a more focused and sensitive approach to the study of gene expression than gene chips, while offering higher throughput than more established approaches to QRT-PCR. We have now evaluated LDAs as a means of determining the expression of multiple genes simultaneously in human tissues and cells.ResultsComparisons between LDAs reveal low variability, with correlation coefficients close to 1. By performing 2-fold and 10-fold serial dilutions of cDNA samples in the LDAs we determined a clear linear relationship between the gene expression data points over 5 orders of magnitude. We also showed that it is possible to use LDAs to accurately and quantitatively detect 2-fold changes in target copy number as well as measuring genes that are expressed with low and high copy numbers in the range of 1 × 102 – 1 × 106 copies. Furthermore, the data generated by the LDA from a cell based pharmacological study were comparable to data generated by conventional QRT-PCR.ConclusionLDAs represent a valuable new approach for sensitive and quantitative gene expression profiling.

Effects of peptidase inhibition on angiotensin receptor agonist and antagonist potency in rabbit isolated thoracic aorta

1 Experiments were performed with peptidase inhibitors on rabbit aortic strip preparations, to determine whether endogenous peptidase activity can influence the potency estimates for angiotensin receptor agonists and antagonists in this tissue. 2 Angiotensin II (A II) and angiotensin III (A III) both induced concentration‐related contractions of rabbit aortic strip preparations. A III was approximately 38 fold less potent than A II, and the gradient of the A III concentration‐response curve (1.00 ±0.04) was significantly more shallow than that (1.76 ± 0.05) of the A II curve. 3 Neither the aminopeptidase‐A and ‐M inhibitor, amastatin, nor the aminopeptidase‐B and ‐M inhibitor, bestatin, affected the potency of, or the maximum response to, A II. In contrast, the potency of A III was increased by both amastatin and bestatin. Amastatin had the most marked effect and at 10 μm caused approximately a 12 fold increase in the potency of A III (EC50 values, 102 nm and 8.6 nm in the absence and presence of amastatin, respectively), and also significantly steepened the gradient of the A III concentration‐response curve. Amastatin did not affect the position or shape of the concentration‐response curve to the α1‐adrenoceptor agonist, phenylephrine. Finally, the carboxypeptidase‐N inhibitor, d‐l‐mercaptomethyl‐3‐guanidine‐ethylpropanoic acid (MERGETPA) did not change the position or shape of the concentration‐response curves to either A II or A III. 4 In the presence of amastatin, the potency of the peptide angiotensin receptor antagonist, Ile7‐A III (100 nm − 1μm), was increased approximately 13 fold (pA2, with A II as the agonist, 7.0 ± 0.1 and 8.1 ± 0.1, in the absence and presence of amastatin, respectively). However, the potency of the non‐peptide angiotensin receptor antagonist, DuP 753 (30–300 nm), was little affected by amastatin (pA2, 8.2 ± 0.1 and 8.1 ± 0.1 in the absence and presence of amastatin, respectively). 5 The results of this study suggest that endogenous aminopeptidase activity in the rabbit thoracic aorta can profoundly affect estimates of the potency of peptide angiotensin receptor agonists and antagonists. A suitable aminopeptidase inhibitor should therefore be included in studies, using this tissue, which aim to classify angiotensin receptor subtype(s) based on the rank order of peptide angiotensin receptor agonist and/or antagonist potencies.

Characterization of three non-peptide endothelin receptor ligands using human cloned ETA and ETB receptors.

1 A number of putative endothelin (ET) receptor ligands were synthesized with a view to assessing their relative affinity for human recombinant ET receptors. 2 Human (h) and endothelin ETA and ETB receptor open reading frames were cloned by reverse transcription‐polymerase chain reaction into the mammalian expression vector pcDNA1 and stable cell lines were created by transfection of Chinese hamster ovary cells. 3 Scatchard analyses of saturation isotherms for the specific binding of [125I]‐endothelin‐1 ([125I]‐ET‐1) to membranes, prepared from Chinese hamster ovary cells transfected with hETA or hETB receptors, yielded values for equilibrium dissociation constants (Kd) of 20.5 ± 1.8 pM and 25.5 ± 5.5 pM, respectively. Hill coefficients did not differ significantly from unity, suggesting binding to homogeneous, non‐interacting receptor populations. 4 Pharmacological characterization of the transfected hETA and hETB receptors was undertaken by measuring the relative abilities of ETA and ETB receptor‐selective peptide ligands to inhibit binding of [125I]‐ET‐1. For interaction with hETA receptors, the relative order of potency was ET‐1 > ET‐3 = FR139317 = BQ123 > [Ala1,3,11,15]‐ET‐1 = sarafotoxin S6c (S6c). In contrast, the relative order of potency, at hETB receptors, was ET‐1 = ET‐3 = [Ala1,3,11,15]‐ET‐1 = S6c >> FR139317 = BQ123. 5 The novel non‐peptide ligands, Ro 46–2005, SB 209670 and BMS 182874, were found to inhibit [125I]‐ET‐1 binding to human recombinant ETA and ETB receptors. At hETA receptors, the calculated pIC50 values were 6.7 (Ro 46–2005), 8.7 (SB 209670) and 5.8 (BMS 182874), while at hETB receptors, the corresponding pIC50 values were 6.8, 7.5 and < 5, respectively. 6 In conclusion, we have characterized the pharmacology of human cloned ETA and ETB receptors and used these in membrane binding assays to determine the affinity and selectivity of three structurally‐diverse non‐peptide ET receptor ligands. SB 209670 is, to date, the highest affinity non‐peptide ligand to be described for ET receptors. As such, it may prove to be a valuable tool in further examination of the physiological and pathophysiological roles of endothelins.

Modulation of tachyphylaxis to angiotensin II in rabbit isolated aorta by the angiotensin AT1 receptor antagonist, losartan.

In rabbit aortic strip preparations, angiotensin II (AII) concentration-contractile response curves (0.3-100 nM) were highly reproducible. However, tachyphylaxis to the contractile response elicited by AII could be induced by repeated exposure to a supramaximal concentration (10 microM) of the peptide. In contrast, a correspondingly supramaximal concentration of the alpha 1-adrenoceptor agonist, phenylephrine (1 mM), did not cause the tissue to become tachyphylactic to phenylephrine. Furthermore, phenylephrine responses were unaffected in tissues previously made tachyphylactic to AII. When the non-peptide, competitive angiotensin AT1 receptor antagonist, losartan (300 nM), was administered before each supramaximal AII challenge, tachyphylaxis did not subsequently occur. Additionally, in tissues made tachyphylactic to AII, subsequent incubation with losartan (300 nM) reversed the AII tachyphylaxis. Thus, losartan may prevent the loss of contractility by preventing AII from interacting with its receptor in a manner which induces tachyphylaxis. However, since losartan can also completely reverse the loss of contractility, it appears capable of restoring the AT1 receptor to a state which allows subsequently administered AII to fully activate the contractile pathway.

Target validation using human tissue: from gene expression to function

Abstract Arguably, one of the main reasons for the pharmaceutical industrys current poor performance in bringing successful new drugs to market is its over-reliance on animal data in the discovery and development processes. We propose that more use should be made of donated human tissues in order to identify and validate drug targets and to discover and develop novel drugs to act upon them. We review the ways that human tissues may be used for this purpose, from the measurement of gene expression, through determination of specific protein abundance, to functional evaluation, both at the level of tissue and specific cell type. The proposal is not simply theoretical, as we provide a specific case history to illustrate these points.