Pharmacokinetic and pharmacodynamic properties of SOL1: a novel dual inhibitor of neutral endopeptidase and endothelin converting enzyme.

AIMS: The pharmacological profile of the novel putative neutral endopeptidase (NEP) and endothelin converting enzyme (ECE) inhibitor SOL1 was examined. MAIN METHODS: The enzyme inhibitory profile of SOL1 was established in vitro. The pharmacokinetic and pharmacodynamic profile was determined in rodents in vivo. KEY FINDINGS: In vitro, at neutral pH, 10 µM SOL1 inhibited NEP-1, NEP-2, and ECE-1 by 99%, 94% and 75%, respectively. The IC(50)s were 25, 25 and 3200 nmol/L, respectively. In anesthetized rats, SOL1 inhibited blood pressure (BP) responses to big-ET-1 and ET-1(1-31) with ED(50)s of 1.9 and 0.03 mg/kg, corresponding to plasma EC(50)s of 4.6 and 0.1 µmol/L, respectively. Pharmacokinetics of SOL1 were examined after single injections in mice and rats. In these species, the estimated clearance of SOL1 varied between 5 and 9 ml/kg.min and T(1/2) between 20 and 60 min. Steady state kinetics of SOL1 were examined after continuous s.c. infusions of SOL1 for 3 weeks at 50mg/kg.day in DOCA-salt hypertensive rats. This treatment lowered BP by 22 mmHg. Steady state concentrations of SOL1 in plasma were 3.9 µmol/L. In heart, lung, and kidney the concentrations of SOL1 were 0.4, 1.8, and 20.5 µmol/kg, respectively. About 63% of the daily dose was retrieved unaltered in the urine. SIGNIFICANCE: These data indicate that SOL1 is primarily a NEP inhibitor in vitro as well as in vivo. Given the preferential renal accumulation and renal clearance of SOL1 additional ECE-1 inhibition in the kidney may have contributed to its chronic BP lowering effects in the DOCA-salt hypertensive rat model.

Impaired cardiac functional reserve in type 2 diabetic db/db mice is associated with metabolic, but not structural, remodelling.

AIM: To identify the initial alterations in myocardial tissue associated with the early signs of diabetic cardiac haemodynamic dysfunction, we monitored changes in cardiac function, structural remodelling and gene expression in hearts of type 2 diabetic db/db mice. METHODS: Cardiac dimensions and function were determined echocardiographically at 8, 12, 16 and 18 weeks of age. Left ventricular pressure characteristics were measured at 18 weeks under baseline conditions and upon dobutamine infusion. RESULTS: The db/db mice were severely diabetic already at 8 weeks after birth, showing elevated fasting blood glucose levels and albuminuria. Nevertheless, echocardiography revealed no significant changes in cardiac function up to 18 weeks of age. At 18 weeks of age, left ventricular pressure characteristics were not significantly different at baseline between diabetic and control mice. However, dobutamine stress test revealed significantly attenuated cardiac inotropic and lusitropic responses in db/db mice. Post-mortem cardiac tissue analyses showed minor structural remodelling and no significant changes in gene expression levels of the sarcoplasmic reticulum calcium ATPase (SERCA2a) or beta1-adrenoceptor (beta1-AR). Moreover, the phosphorylation state of known contractile protein targets of protein kinase A (PKA) was not altered, indicating unaffected cardiac beta-adrenergic signalling activity in diabetic animals. By contrast, the substantially increased expression of uncoupling protein-3 (UCP3) and angiopoietin-like-4 (Angptl4), along with decreased phosphorylation of AMP-activated protein kinase (AMPK) in the diabetic heart, is indicative of marked changes in cardiac metabolism. CONCLUSION: db/db mice show impaired cardiac functional reserve capacity during maximal beta-adrenergic stimulation which is associated with unfavourable changes in cardiac energy metabolism.

Acute autonomic effects of vitamins and fats in male smokers.

BACKGROUND/OBJECTIVES: Vitamins can help improve cardiovascular control. In contrast, smoking works in the opposite fashion, reducing the baroreflex control of heart rate (HR) possibly via oxidative stress. High-fat challenges also impair cardiovascular regulation. Whether vitamins have acute beneficial effects on the baroreflex control of HR in smokers is unclear. SUBJECTS/METHODS: A randomized, placebo-controlled crossover study in 30 male smokers (34.2+/-6.9 years). Interventions were: (1) moderate (vitamin C (300 mg) and E (75 IU) and folic acid (1 mg)); (2) high doses of vitamins (vitamin C (2 g) and E (800 IU), and folic acid (5 mg)); or, (3) placebo. Vitamins were ingested with cream (a high-fat challenge) or milk (low-fat control). Four hours later, blood was withdrawn and radial pulse wave forms recorded via tonometry. Spontaneous beat-to-beat variations in HR and systolic blood pressure (SBP) were analysed by spectral analysis techniques and sympathovagal control of HR and baroreflex sensitivity (BRS) were assessed. RESULTS: High doses of vitamins increased plasma vitamin C, E and folic acid levels (P<0.05) with no change in SBP, HR or BRS (P>0.05, analysis of variance). Plasma vitamin levels did not correlate with any cardiovascular parameters. Moderate vitamins increased the vagal control of HR (+23%; P<0.05) and cream led to small increases (P<0.05) in SBP (+2 mm Hg) and HR (+2 beats min(-1)) with no change in BRS. CONCLUSIONS: In male smokers, circulating antioxidants had no effect on BRS and minor effects on the cardiovascular system were seen following acute fat and vitamin ingestion.

Renal medullary effects of transient prehypertensive treatment in young spontaneously hypertensive rats.

AIM: Transient angiotensin II receptor blockade (ARB) leads to prolonged blood pressure (BP) lowering, but the underlying mechanism remains uncertain. Long-term BP control is regulated by the medullary microcirculation with the pericyte as contractile cell. We hypothesize that the prolonged BP effect is caused by increased medullary blood flow (MBF) associated with structural alterations based on reduced medullary pericyte number. METHODS: Four-week-old spontaneously hypertensive rats (SHR) were treated for 4 weeks with losartan (SHR-Los: 20 mg kg(-1) day(-1)), hydralazine (SHR-Hyd: 15 mg kg(-1) day(-1)), losartan and pan-caspase inhibitor zVAD (SHR-Los + 1 mg kg(-1) day(-1) zVAD), losartan and glycogen synthase kinase-3beta (GSK) inhibitor valproate (SHR-Los + 10 mg kg(-1) day(-1) Val) or placebo. BP, MBF and pericyte number were determined under and after treatment (8 and 12 weeks). Apoptotic pericytes were determined with alpha-actin and TUNEL double staining. Sodium concentration was determined in renal medulla and urine. RESULTS: Antihypertensive treatment equipotently reduced BP at 8 weeks of age. After drug withdrawal (12 weeks of age) BP reduction was restricted to SHR-Los (SHR-Los: 153 +/- 5, SHR-Hyd: 177 +/- 2, SHR: 184 +/- 3 mmHg). Simultaneously, MBF was increased and pericyte number reduced, while medullary and urinary sodium concentration increased. Transient ARB in combination with zVAD or valproate resulted in more medullary pericytes and higher BP (SHR-Los/zVAD: 164 +/- 7; SHR-Los/Val: 168 +/- 6 mmHg) compared with transient ARB alone. CONCLUSION: After drug withdrawal, transient ARB leads to increased MBF and is associated with a reduction in medullary pericytes. This may be associated with pericyte apoptosis as anti-apoptosis during transient ARB increases pericyte number and BP.

Oestrogen modulates cardiac ischaemic remodelling through oestrogen receptor-specific mechanisms.

AIM: Observational and clinical studies suggest different responses upon sex hormone replacement therapy in ischaemic heart disease. Few studies, however, have examined the impact of oestrogen receptor-dependent mechanisms on the extent of injury after myocardial infarction (MI). Therefore, we set out to evaluate the effect of oestrogen (E2) replacement on infarct size and remodelling, and the respective role of the oestrogen receptors (ER)alpha and -beta in this process, using ERalpha- and ERbeta-deficient mice. METHODS: Wild type (WT) (ERalpha(+/+) and ERbeta(+/+)), ERalpha-deficient (ERalpha(-/-)) and ERbeta-deficient (ERbeta(-/-)) mice were ovariectomized and subsequently supplemented with E2 or placebo using subcutaneous 60-day release pellets. MI was induced by left coronary artery ligation. Two weeks following MI, haemodynamic function was assessed and infarct size was determined. RESULTS: There was no significant difference in infarct size between E2- or placebo-treated WT (ERalpha(+/+) and ERbeta(+/+)) mice. Surprisingly, E2 treatment did result in smaller infarct sizes in ERalpha(-/-) mice, but increased the infarct size in ERbeta(-/-) mice. Increase of the left ventricular mass post-MI was significantly larger in the E2-treated ERalpha(-/-) animals compared with placebo-treated animals. E2 treatment also significantly increased post-MI mortality in ERalpha(+/+), ERbeta(+/+) and ERalpha(-/-) animals, but not in ERbeta(-/-) mice. CONCLUSIONS: Although E2 modulates the infarct size in ERalpha(-/-), it also appears to be responsible for the higher mortality following MI. ERbeta appears to be the receptor involved in the modulating effects of E2 in the infarcted heart.

Specific and sustained down-regulation of genes involved in fatty acid metabolism is not a hallmark of progression to cardiac failure in mice.

Preferential and specific down-regulation of genes involved in fatty acid (FA) uptake and metabolism is considered a hallmark of severe hypertrophic remodeling and progression to cardiac failure. Therefore, we investigated the time course of changes in cardiac metabolic gene expression (1) in mice subjected to regional myocardial infarction (MI) for 4 days, 1 month, or 3 months and (2) in mice overexpressing calcineurin (Cn) which initially develop concentric hypertrophy progressing after the age of 4 weeks to dilated cardiomyopathy and failure. In both models, hypertrophy was characterized by increased expression of beta-myosin heavy chain protein and atrial natriuretic factor mRNA, indicative of marked structural remodeling. Fractional shortening progressively decreased from 31% to 15.1% and 3.7% 1 and 3 months after MI, respectively. One month post-MI, the expression of several metabolic genes, i.e., acyl-CoA synthetase (-50%), muscle-type carnitine palmitoyl transferase 1 (-37%) and citrate synthase (-28%), was significantly reduced in the surviving myocardium. Despite overt signs of cardiac failure 3 months post-MI, the expression of these genes had returned to normal levels. In hearts of both 4- and 6-week-old Cn mice, genes involved in both FA and glucose metabolism and mitochondrial citrate synthase were down-regulated, reflecting an overall decline in metabolic gene expression, rather than a specific and preferential down-regulation of genes involved in FA uptake and metabolism. These findings challenge the concept that specific and sustained down-regulation of genes involved in FA uptake and metabolism represents a hallmark of the development of cardiac hypertrophy and progression to failure.

Chronic moderate hypoxia during in ovo development alters arterial reactivity in chickens.

We previously observed arterial sympathetic hyperinnervation and endothelial dysfunction in the chicken embryo after exposure to chronic hypoxia. We now investigate whether changes in arterial properties could also be observed at 14-15 weeks of life. Eggs of White Leghorn chicken were incubated under normoxic or moderately hypoxic (15% O2 from days 6-19 of a 21-day incubation) conditions. Experiments were performed at 14-15 weeks of life under standard conditions (Hm: males exposed to hypoxia; Hf: females exposed to hypoxia; Nm: males exposed to normoxia; Nf: females exposed to normoxia). Body weight at hatching and at 14-15 weeks was not affected by in ovo exposure to hypoxia. Mean arterial pressure and heart rate were not significantly altered by chronic in ovo hypoxia. However, isolated femoral arteries were more sensitive to electrical stimulation (frequency in Hz of half-maximal contraction, Hm: 1.62+/-0.33, Hf: 1.92+/-0.88, Nm: 2.49+/-0.49, Nf: 2.83+/-0.31) and pharmacological stimulation of peri-arterial sympathetic nerves (contraction in N/m in response to tyramine: Hm: 5.27+/-0.85, Hf: 4.10+/-0.9, Nm: 2.26+/-0.67, Nf: 3.65+/-0.51, p=0.07) after in ovo hypoxia. In side branches of the femoral artery, the effect of NO synthase blockade with L-NAME on contraction (in N/m) in response to high K+ (Hm: 0.35+/-0.91, Hf: 1.29+/-0.36, Nm: 2.88+/-0.19, Nf: 2.79+/-0.58) and on the sensitivity to acetylcholine (DeltapD2, H: 0.32+/-0.11, N: 0.62+/-0.05) was reduced after in ovo hypoxia. The present study shows that exposure to chronic moderate hypoxia during development affects the contractile and relaxing arterial responses of 14- to 15-week-old chickens. Although hypoxia did not lead to changes in blood pressure at this age, the observed effects on arterial sympathetic and endothelial function may represent early signs of future cardiovascular abnormalities.