Kenneth Lyle Clark

Quantitative mRNA expression profiling of ACE 2, a novel homologue of angiotensin converting enzyme

ACE 2, a novel homologue of angiotensin converting enzyme, has recently been identified. This study used QRT‐PCR to quantitatively map the transcriptional expression profile of ACE 2 (and the two isoforms of ACE) in 72 human tissues. While confirming that ACE 2 expression is high in renal and cardiovascular tissues, the novel observation has been made that ACE 2 shows comparably high levels of expression in the gastrointestinal system, in particular in ileum, duodenum, jejunum, caecum and colon. Therefore, in probing the functional significance of this novel peptidase, some consideration should be given to a role in gastrointestinal physiology and pathophysiology.

Cardiovascular pharmacology of the adenosine A1/A2-receptor agonist AMP 579: coronary hemodynamic and cardioprotective effects in the canine myocardium.

The hemodynamic and cardioprotective properties of the novel adenosine A1/A2 receptor agonist AMP 579 (IS-[1a,2b,3b,4a(S*)]-4-[7-[[1-[(3-chloro-2-thienyl)methyl]propylamino]- 3H-imidazo[4,5-b]pyridin-3-yl]-N-ethyl-2,3-dihydroxy cyclopentanecarboxamide) were studied in two canine models designed to simulate (a) mild single-vessel coronary artery disease, and (b) myocardial ischemia/reperfusion injury. In the first model, a moderate stenosis was placed on the left circumflex coronary artery (LCCA), and the effects of AMP 579 on regional myocardial blood flow were assessed. AMP 579, 10 micrograms/kg/min, i.v., for 10 min, induced coronary dilation without causing endocardial steal. In the model of ischemia/reperfusion injury (60 min LCCA occlusion/5 h reperfusion), AMP 579, 10 micrograms/kg/min, i.v., administered for 15 min before ischemia significantly decreased myocardial infarct size. Control infarct size to area at risk (IS/AAR) equaled 34 +/- 3% (n = 9); IS/AAR for AMP 579-treated dogs equaled 16 +/- 4% (n = 9). Preconditioning (5 min LCCA occlusion + 10 min reperfusion) immediately before the 60-min LCCA occlusion also resulted in a marked decrease in IS/AAR: 9 +/- 3% (n = 6). The selective A1 agonist CPA reduced infarct size when administered at 3 micrograms/kg/min, i.v., for 15 min before LCCA occlusion: IS/AAR = 11 +/- 3% (n = 5). Pretreatment of animals with the adenosine-receptor antagonist 8-SPT, 10 mg/kg, i.v., attenuated the myocardial protective effects associated with preconditioning, CPA, and AMP 579, resulting in IS/AAR values of 28 +/- 7% (n = 7), 28 +/- 4% (n = 8), and 26 +/- 3% (n = 8), respectively. The ability of 8-SPT to block the cardioprotective effects suggests that these effects were mediated through an interaction with adenosine receptors. These experimental results indicate that AMP 579 is an effective coronary vasodilator, which also can protect the heart from ischemic injury. Thus AMP 579 has the potential to be useful in cardiovascular therapeutics.

Role of angiotensin AT1 and AT2 receptors in mediating the renal effects of angiotensin II in the anaesthetized dog

1 Experiments were performed using the selective AT1 receptor antagonist, GR117289, and the selective AT2 receptor antagonist, PD123177, to assess the relative importance of AT1 versus AT2 receptors in mediating the renal effects of angiotensin II (AII) in vivo, in salt‐replete pentobarbitone‐anaesthetized dogs. 2 The AT1 receptor antagonist, GR117289 (0.5 mg kg−1 + 1 μg kg−1 min−1, i.v.), caused renal vasodilatation, characterized by a mean increase of 21 ± 5% in renal blood flow, 45 min post‐dose. GR117289 also caused a fall in mean blood pressure (12 ± 4%), but despite this, sodium and urine excretion were not reduced. Indeed, there was a tendency for urine output and sodium excretion to increase, although the changes were not statistically significant. GR117289 caused a reduction in plasma aldosterone levels (−35 ± 16%) 45 min post‐dose, despite increasing plasma renin activity (+ 173 ± 42%). In contrast to GR117289, the AT2 receptor antagonist, PD123177 (20 μg kg−1 min−1 intra‐renal artery; i.r.a.) caused no significant change in blood pressure, renal blood flow, or sodium and urine excretion, indicating that the renal effects of endogenous AII in these salt‐replete animals are mediated predominantly by AT1 receptors. 3 Intra‐renal artery infusion of AII (1–300 ng kg−1 min−1) caused dose‐related renal vasoconstriction, and decreases in urine output, sodium excretion, fractional excretion of sodium, and glomerular filtration rate (GFR). The AT1 receptor antagonist, GR117289 (0.5 mg kg−1 + 1 μg kg−1 min−1, i.v.) antagonized these renal effects of AII, causing 15–38 fold rightward displacements of mean dose‐response curves for these parameters. In contrast, PD123177 (20 μg kg−1 min−1, i.r.a.) failed to antagonize the renal haemodynamic and excretory effects of lower doses of AII (1–10 ng kg−1 min−1, i.r.a.). However, at higher doses of AII (30–300 ng kg−1 min−1, i.r.a.), while PD123177 still failed to antagonize the effects of the peptide on urine output, sodium excretion and GFR, it did cause a small, but significant, degree of inhibition of AII‐induced renal vasoconstriction. In addition, at a higher dose (50 μg kg−1 min−1, i.r.a.), PD123177 caused a greater degree of antagonism of AII‐induced renal vasoconstriction, while renal excretory responses to AII remained unaffected. 4 This study shows that the renal haemodynamic and excretory effects of AII in salt‐replete anaesthetized dogs are mainly mediated by angiotensin AT1 receptors. However, the inhibitory effect of PD123177 on renal vasoconstrictor responses to high doses of AII, raises the possibility that functionally important AT2 receptors are present in the canine renal vasculature.

Characterization of endothelin receptors in rat renal artery in vitro

1 The aim of this study was to investigate the function and characteristics of endothelin receptors in rat main branch renal artery in vitro. 2 Endothelin(ET)‐1 (mean EC50 = 9.8 nm) was approximately 12 fold more potent than ET‐3 (mean EC50 = 120 nm) as a contractile agonist and produced a greater maximum response. In contrast, neither of the ETB receptor‐selective agonists, alanine1,13,11,15] ET‐1 nor sarafotoxin S6c, (0.1 nm‐1 μm), induced any contractile effect, or any relaxant effect in endothelium‐intact preparations pre‐contracted with the thromboxane A2 mimetic, U‐46619. Sarafotoxin S6c (30 nm) also failed to induce any further contraction in tissues pre‐contracted with an EC50 concentration of ET‐1. 3 The ETA receptor‐selective antagonist, BQ123, behaved as a weak and variable antagonist of the contractile effects of ET‐1 (mean pA2 estimates in the range 5.8–6.3). In contrast, BQ123 antagonized ET‐3 with a potency (mean pA2 = 7.6) consistent with its affinity for ETA receptors. Co‐incubation of BQ123 (3 μm) with the putative ETB receptor‐selective antagonist, IRL1038 (10 μm), produced no greater antagonism of ET‐1 responses than was induced by BQ123 (3 μm) alone. 4 In conclusion, ETB receptors do not appear to be present in rat main branch renal artery. The contractile effects of ET‐3 in this tissue seem to be mediated by ETA receptors. While ETA receptors partly mediate the contractile effects of ET‐1, these data raise the possibility that a population of novel BQ123‐insensitive endothelin receptors may also contribute to this response.

Do renal tubular dopamine receptors mediate dopamine-induced diuresis in the anesthetized cat?

The aim of this study was to investigate whether the pentobarbitone anesthetized cat is a suitable preparation in which to characterize renal tubular dopamine receptors. Intravenous infusion of dopamine (10–100 μg/kg/min) resulted in a dose-related increase in mean blood pressure (MBP). urine output, sodium excretion (UNa V), and fractional sodium excretion (FENa). This diuretic effect occurred despite little change in glomerular filtration rate, suggesting that it is a consequence of decreased tubular reabsorption. Dopamine (10–100 μg/kg/min, i.v.) also induced marked dose-renal blood flow was not significantly reduced. The increases in MBP, urine output, UNa V, and FENa induced by dopamine (10–100 μg/kg/min, i.v.), were unaffected by pretreatment of cats with either the selective dopamine DA1 or DA2 receptor antagonists, SCH 23390 (30 μg/kg, i.v.), or domperidone (100 μg/kg, i.v.), respectively. In contrast, pretreatment of cats with the nonselective α-adrenoceptor antagonist, phentolamine (1 mg/kg, i.v.) prevented dopamine-induced increases in urine output and MBP. Infusion of the selective dopamine DA1 receptor agonist fenoldopam (0.01–10 μg/kg/min) into the left renal artery failed to increase left renal vascular conductance, or left kidney urine output, UNa V, or FENa. In conclusion, this study provides no evidence for the involvement of renal tubular dopamine receptors in dopamine-induced diuresis in anesthetized cats. Rather, the diuretic effect appears to be linked to stimulation of α-andrenoceptors.

Induction of heat shock protein 70 by herbimycin A and cyclopentenone prostaglandins in smooth muscle cells

Abstract This study characterizes Hsp70 induction in human smooth muscle cells (SMC) by herbimycin A and cyclopentenone prostaglandins. The magnitude of Hsp70 induction by cyclopentenone prostaglandins was 8- to 10-fold higher than induction by herbimycin A. Hsp70 induction by Δ12PGJ2 was first observed at 10 μM, rose to 4000–5000 ng/mL within one log unit and a maximum response was not observed; concentrations of Δ12PGJ2 higher than 30 μM were toxic to the cells. A maximum response with herbimycin A (500 ng/mL) was reached at 0.05 μM and maintained to 1 μM without toxicity. Both, Δ12PGJ2 and herbimycin A, were inhibited by dithiothreitol (DTT, 100 μM) at lower concentrations and became less sensitive to inhibition at higher concentrations. Hsp70 induction after incubation of SMC with Δ12PGJ2 followed by addition of herbimycin A was significantly higher than Hsp70 induction after incubation with herbimycin A followed by addition of Δ12PGJ2. When cells were incubated with [3H]-PGJ2, followed by protein denaturation, substantial radioactivity remained protein-bound suggesting that the prostaglandin must be covalently bound. Covalent binding was largely insensitive to DTT. Maximal Hsp70 induction was observed after 5 minutes of exposure of the cells to herbimycin A followed by a 20 hour recovery period in agent-free medium. Cells required 3–4 hours of exposure to Δ12PGJ2 followed by a 20 hour recovery period in order to see high Hsp70 induction. Binding of the heat shock factor (HSF) to the heat shock element (HSE) in the presence of herbimycin A or Δ12PGJ2, and the effects of DTT, mirrored the results of Hsp70 induction. The results suggest that probable differences between the 2 agents are at the level of the signal transduction prior to HSF activation.

Does tumor necrosis factor-α (TNF-α) contribute to myocardial reperfusion injury in anesthetized rats?

Abstract 1. This study examined the potential role of tumor necrosis factor-alpha (TNF-α) in myocardial ischemia-reperfusion injury using an anesthetized rat model of myocardial infarction. 2. The key endpoints were infarct size and serum TNF-α levels (measured by a specific ELISA technique). 3. Three groups of rats were studied: vehicle controls (n=6); positive controls for infarct size reduction (ischemic preconditioning; n=6); and a group treated with the selective inhibitor of PDE-IV and TNF-α production, rolipram (1mg/kg IV 10-min prereperfusion+1 μg/kg per minute through 1-hr reperfusion, n=6). 4. Baseline preischemia levels of serum TNF-α were low (∼0.1 nM) and showed a trend for further reduction in all treatment groups at 1 min and 3 hr into the postischemia reperfusion period. 5. Infarct size (68±2% of the ischemic area in controls) was significantly reduced (41% decrease) by preconditioning, but was unchanged in rolipram-treated animals. 6. Collectively, these data argue against an important role for TNF-α in lethal reperfusion injury in this rat model of myocardial infarction.

A comparison of the characteristics of angiotensin receptors in the renal and mesenteric vascular beds of the anesthetized cat

Experiments were performed in anesthetized cats to compare the characteristics of angiotensin receptors in the renal and mesenteric vascular beds. Injection of either angiotensin II (Ang II; 0.3–30 ng) or angiotensin HI (Ang HI; 0.3–30 ng) directly into the superior mesenteric or renal artery caused dose-related, reproducible reductions in mesenteric and renal blood flow, respectively. Ang II and Ang III were equipotent as vasoconstrictors in both vascular beds. The peptide angiotensin receptor antagonists saralasin and IIe7-Ang III (1 μg/kg/min intravenously, i.v.) and the nonpeptide angiotensin receptor antagonist DuP 753 (3 mg/kg plus 20 μg/kg/min i.v.) caused a rightward displacement of dose-response curves to Ang II or Ang III in both the mesenteric and renal vasculature. Vasoconstrictor responses to Ang II or Ang HI in either vascular bed were blocked to similar extents by each antagonist. In separate cats, the dose of the antagonists required to cause a 10-fold rightward displacement of the Ang II dose-response curve (DR10) in both vascular beds was determined. The DR10 values indicated that the potency of each antagonist was similar in the renal and mesenteric vascular beds. These results provide no evidence to suggest that angiotensin receptors mediating vasoconstriction in the renal and mesenteric vasculature have different characteristics.

From virtual to clinical: The discovery of PGN-1531, a novel antagonist of the prostanoid EP4 receptor.

In this Letter, we present the results of a hit-finding and lead optimization programme against the EP4 receptor (EP4R). In a short time period, we were able to discover five structurally diverse series of hit compounds using a combination of virtual screening methods. The most favoured hit, compound 6, was demonstrated to be a competitive antagonist of the EP4R. Compound 73 was identified following several rounds of optimization, which centred on improving both the primary EP4R affinity and selectivity against the related EP2R as well as the aqueous solubility. This work culminated in the preparation of PGN-1531, the sodium salt of 73, which showed a marked improvement in solubility (>10 mg/mL). PGN-1531 is a potent and selective antagonist at EP4Rs in vitro and in vivo, with the potential to alleviate the symptoms of migraine that result from cerebral vasodilatation.

Potential of adenosine receptor agonists for the prevention and treatment of coronary artery disease and acute myocardial infarction

Adenosine is an ubiquitously produced autocoid which mediates its effects via four receptor subtypes (A1, A2A, A2B and A3) which show a relatively widespread tissue distribution. Adenosine itself is upregulated in vascular tissue in response to hypoxia and induces vasodilation as a homeostatic response. The vasodilator effects of adenosine are exploited clinically, where it is used as a pharmacological vasodilator during stress testing. More recently, it has become clear that adenosine and adenosine agonists possess significant potential for cardioprotection. Thus, given prior to an ischaemic insult, adenosine and adenosine A1 receptor agonists can reduce infarct size, reduce arrhythmia and improve post-ischaemic cardiac function. In addition, when given during myocardial ischaemia just prior to reperfusion, adenosine and adenosine A2A receptor agonists can inhibit neutrophil adhesion, activation and infiltration into post-ischaemic myocardium, thereby inhibiting lethal reperfusion injury and further salvaging myocardial tissue. There is also some evidence that stimulation of adenosine A3 receptors can protect myocardium. The realisation that adenosine analogues possess cardioprotective activity has stimulated research to identify subtype selective analogues with the optimal profile for use in acute myocardial infarction (MI) and cardiac surgery. Several compounds have reached the stage of early clinical development and hold significant promise as future therapies. In addition to direct cardioprotective effects, adenosine A1 selective agonists may also indirectly benefit the heart via their metabolic effects. Thus, adenosine A1 receptor stimulation results in a marked suppression of lipolysis in adipose tissue, which in turn leads to a reduction in the levels of circulating free fatty acids, triglycerides and very low density lipoproteins. Triglycerides are now accepted as an independent risk factor for mortality from coronary artery disease. Thus, the potential exists for the development of orally-active selective A1 receptor agonists for the treatment of hypertriglyceridaemic patients to reduce the incidence of primary or secondary coronary events. Progress is being made in the design of such A1 selective agonists.Therefore, the next decade should see advances in the therapeutic application of adenosine agonists in the porphylactic treatment of coronary artery disease and in acute MI.