1472 Aldosterone and pressor responses to torcetrapib MJ Forrest et al Figure 8 Torcetrapib released aldosterone from isolated rat adrenal cells. Aldosterone release was measured from primary adrenocortical cells isolated from rat adrenal glands. Cells (B200 000 per sample) were incubated with angiotensin II (0.1 mM), torcetrapib (1–30 mM), anacetrapib (0.1–10 mM) or appropriate vehicle for 2 h at 37 1C. Following centrifugation, supernatants were assayed for aldosterone by an enzyme-linked immunoassay. Each bar represents the mean and vertical lines represent the s.e. for four samples. Several lines of evidence indicate a relationship between elevated plasma aldosterone concentration, hypertension and increased cardiovascular events (Rocha and Funder, 2002; Vasan et al., 2004; Connell and Davies, 2005). The relevance of the acute blood pressure elevation and increase in plasma aldosterone in response to torcetrapib reported herein to the blood pressure elevation and adverse cardiovascular events described in the ILLUMINATE trial is uncertain. Aldosterone is a major regulator of sodium and water homoeostasis, thus contributing to the maintenance of normal blood volume and blood pressure. Increased aldosterone levels promote in- creased sodium reabsorbtion with subsequent facilitated potassium and hydrogen ion exchange, volume expansion and elevated blood pressure. These classical effects of aldosterone are mediated through binding of aldosterone to the well-studied mineralocorticoid receptor (NR3C2) in epithelial cells, primarily in the distal nephron of the kidney. More recently, it has become appreciated that there are non- classical aspects of aldosterone biology including synthesis at sites other than the adrenal cortex; the presence of miner- alocorticoid receptors in non-epithelial tissues including heart, brain and blood vessels; and actions of aldosterone via non-genomic mechanisms (Rocha and Funder, 2002; Connell and Davies, 2005). The strongest evidence that aldosterone is a contributor to adverse cardiovascular events comes from clinical trials with mineralocorticoid receptor antagonists. Two key studies, Randomized Aldactone Evaluation Study (Pitt et al., 1999) British Journal of Pharmacology (2008) 154 1465–1473 and Eplerenone Post-acute myocardial infarction Heart failure Efficacy and Survival study (Pitt et al., 2003) demon- strated benefits of mineralocorticoid receptor blockade in patients with heart failure. Interestingly, in both studies, benefits were observed shortly after randomization, suggest- ing that aldosterone receptor blockade could be acting to limit cardiac electrical disturbances. Collectively, a considerable body of evidence is available to support the concept that elevated plasma aldosterone concentration is associated with adverse cardiovascular effects and that reducing either the levels of aldosterone or the effects of aldosterone is beneficial. Whether the in- creased incidence of death and adverse cardiovascular events reported in the ILLUMINATE trial were attributable to the reported increase in plasma aldosterone is unknown. The studies reported herein have shown an acute increase in plasma aldosterone in rodents given torcetrapib. We are yet to determine whether aldosterone levels remain elevated with chronic administration of torcetrapib, and we do not know whether this phenomenon is observed across a broad range of species. The mechanism for the acute blood pressure effect of torcetrapib remains to be determined. Torcetrapib did not have a direct action on vascular tone, as it did not contract isolated arterial vascular segments in vitro and did not enhance responses to other vasoactive agents. Torcetrapib also did not affect the vascular endothelial nitric oxide system as demonstrated by a lack of effect on ACh-induced relaxation in isolated vascular preparations. Possible effects of torcetrapib on smaller resistance vessels seem unlikely, given that torcetrapib did not increase pressure in an isolated perfused hindlimb preparation. Possible effects on cardiac output were evaluated using high-frequency ultrasound at the level of the ascending aorta. These studies showed no remarkable change in cardiac output of anaesthetized rats at doses where blood pressure is increased (data not shown). Collectively, these data indicate that the mechanism does not involve a direct effect on the cardiovascular system and is most likely via a hormonal or endogenous mediator system. Pharmacological studies have excluded activation of angiotensin AT1 receptors, endothelin receptors or a1/a2- adrenoceptors. Possible central effects of torcetrapib were excluded because equivalent pressor effects of torcetrapib are present in both normal and pithed anaesthetized rats in which all central pathways are eliminated. Administration of torcetrapib in a variety of animal models evoked an acute increase in blood pressure and in rats it was shown to increase plasma aldosterone and corticosterone levels. Whether either or both of these properties contributed to the adverse cardiovascular events reported in the ILLUMINATE trial is unclear. It is also unclear whether these properties of torcetrapib conspired against determining significant beneficial effects of torcetrapib on HDL and atherosclerotic plaque volume. In atherosclerosis- prone mice, mineralocorticoid receptor blockade with eplerenone not only reduced blood pressure but also significantly reduced atherosclerotic lesion area by 35% (Keidar et al., 2003). Thus, it is possible that elevated aldosterone levels contribute to the development of athero- sclerotic plaque. The question as to whether HDL elevation