Levosimendan alone and in combination with valsartan prevents stroke in Dahl salt-sensitive rats.

The effects of levosimendan on cerebrovascular lesions and mortality were investigated in models of primary and secondary stroke. We aimed to determine whether the effects of levosimendan are comparable to and/or cumulative with those of valsartan, and to investigate whether levosimendan-induced vasodilation has a role in its effects on stroke. In a primary stroke Dahl/Rapp rat model, mortality rates were 70% and 5% for vehicle and levosimendan, respectively. Both stroke incidence (85% vs. 10%, P<0.001) and stroke-associated behavioral deficits (7-point neuroscore: 4.59 vs. 5.96, P<0.001) were worse for vehicle compared to levosimendan. In a secondary stroke model in which levosimendan treatment was started after cerebrovascular incidences were already detected, mean survival times were 15 days with vehicle, 20 days with levosimendan (P=0.025, vs. vehicle), 22 days with valsartan (P=0.001, vs. vehicle), and 31 days with levosimendan plus valsartan (P<0.001, vs. vehicle). The respective survivals were 0%, 16%, 20% and 59%, and the respective incidences of severe lesions were 50%, 67%, 50% and 11%. In this rat model, levosimendan increased blood volume of the cerebral vessels, with significant effects in the microvessels of the cortex (∆R=3.5±0.15 vs. 2.7±0.17ml for vehicle; P=0.001) and hemisphere (∆R=3.2±0.23 vs. 2.6±0.14ml for vehicle; P=0.018). Overall, levosimendan significantly reduced stroke-induced mortality and morbidity, both alone and with valsartan, with apparent cumulative effects, an activity in which the vasodilatory effects of levosimendan have a role.

Caloric restriction ameliorates angiotensin II-induced mitochondrial remodeling and cardiac hypertrophy.

Angiotensin II-induced cardiac damage is associated with oxidative stress-dependent mitochondrial dysfunction. Caloric restriction (CR), a dietary regimen that increases mitochondrial activity and cellular stress resistance, could provide protection. We tested that hypothesis in double transgenic rats harboring human renin and angiotensinogen genes (dTGRs). CR (60% of energy intake for 4 weeks) decreased mortality in dTGRs. CR ameliorated angiotensin II-induced cardiomyocyte hypertrophy, vascular inflammation, cardiac damage and fibrosis, cardiomyocyte apoptosis, and cardiac atrial natriuretic peptide mRNA overexpression. The effects were blood pressure independent and were linked to increased endoplasmic reticulum stress, autophagy, serum adiponectin level, and 5' AMP-activated protein kinase phosphorylation. CR decreased cardiac p38 phosphorylation, nitrotyrosine expression, and serum insulin-like growth factor 1 levels. Mitochondria from dTGR hearts showed clustered mitochondrial patterns, decreased numbers, and volume fractions but increased trans-sectional areas. All of these effects were reduced in CR dTGRs. Mitochondrial proteomic profiling identified 43 dTGR proteins and 42 Sprague-Dawley proteins, of which 29 proteins were in common in response to CR. We identified 7 proteins in CR dTGRs that were not found in control dTGRs. In contrast, 6 mitochondrial proteins were identified from dTGRs that were not detected in any other group. Gene ontology annotations with the Panther protein classification system revealed downregulation of cytoskeletal proteins and enzyme modulators and upregulation of oxidoreductase activity in dTGRs. CR provides powerful, blood pressure-independent, protection against angiotensin II-induced mitochondrial remodeling and cardiac hypertrophy. The findings support the notion of modulating cardiac bioenergetics to ameliorate angiotensin II-induced cardiovascular complications.

Toxicology and safety of COMT inhibitors.

The development of catechol-O-methyltransferase (COMT) inhibitors for the adjunct treatment to levodopa and aromatic L-amino acid decarboxylase (AADC) inhibitors in Parkinson's disease started in the late 1950s. The first-generation inhibitors were associated with toxic properties: they induced convulsions, or they were toxic to the liver. None of them was taken into clinical use. The second-generation inhibitors entacapone and tolcapone have now been in clinical use for over a decade, and some new inhibitors are under development. The main adverse events in the use of entacapone and tolcapone are dopaminergic and dependent of the concomitant use of levodopa, but the symptoms are generally moderate or mild. Among the non-dopaminergic adverse events, diarrhea is the most prominent one induced by both entacapone and tolcapone. In clinical use, entacapone has been safe, but tolcapone is under strict regulations on liver enzyme monitoring, since in the early years, a few hepatotoxicity cases appeared, three of them with fatal outcome. The mechanism behind tolcapone-induced liver toxicity has been evaluated both in vitro and in vivo, but no clear answer exists at the moment. In the regulatory animal studies, both inhibitors have been safe with no reported toxicity. Also nebicapone, the latest of the second-generation inhibitors in clinical trials has shown some liver enzyme elevations in human subjects. New inhibitors with a structure differing from nitrocatechols are under development. No safety concerns have been reported connected to COMT inhibiton as such. COMT knockout mice are fertile without any pathologies due to the total COMT inhibition.

Effect of simvastatin, an established lipid-lowering drug, on pulmonary Chlamydia pneumoniae infection in mice.

The effects of simvastatin treatment on Chlamydia pneumoniae lung infection, inflammation, and serum lipids in mouse model were studied. Simvastatin decreased viable chlamydial counts and increased inflammatory cell infiltrates in the lung tissue, suggesting that simvastatin treatment had both antichlamydial and immunomodulatory effects during an acute C. pneumoniae infection.

Tissue histopathology, clinical chemistry and behaviour of adult Comt-gene-disrupted mice.

Catechol-O-methyltransferase (COMT) enzyme is a widely distributed enzyme that catalyses O-methylation of catecholamines and other compounds having a catechol structure. Because there has been some concern about the consequences of a low COMT activity in the development of oestrogen-dependent cancers and because one of the COMT inhibitors, tolcapone, has caused serious liver injuries in Parkinsonian patients, the histopathology and clinical chemistry of Comt-gene-disrupted mice were studied at the age of 12 months. Owing to the high COMT activities in liver and kidney and the role of COMT in the metabolism of catechol oestrogens, special emphasis was given to the histology of the liver, kidney and oestrogen-dependent organs such as mammary glands and uterus. The mice of both heterozygous and homozygous genotypes appear to be physically healthy and fertile. Diurnal motility rhythm and behaviour in measuring anxiety and depression were equal in all genotypes. At the age of 12 months, the body weight of homozygous mice was 7-9% lower than that of the other groups. This was reflected in histology as a diminished incidence of vacuolation of liver cells (fatty change). Macroscopic pathology and histopathology revealed no abnormal findings in any COMT genotype. The values of some clinical chemistry parameters, such as alkaline phosphatase, alanine aminotransferase, urea, glucose, calcium and proteins, were at a higher level in homozygous animals compared with the wild-type mice. However, all the values remained within the normal physiological range, and the differences in enzyme levels between genotypes were not reflected as histopathological findings in the relevant organs. No changes in haematological parameters or plasma catecholamine concentrations were noted but plasma 3,4-dihydroxyphenylethylene glycol levels were high in COMT null mice. The results suggest that the full or 50% lack of Comt gene as such is not associated with any toxic consequences.

Effects of entacapone and tolcapone on mitochondrial membrane potential.

Catechol-O-methyl transferase (COMT) inhibitors, entacapone and tolcapone, are used as an adjunctive treatment to L-dopa in Parkinson's disease. Based on their catechol structure, both inhibitors are potential uncoupling agents, but only tolcapone shows this effect in vitro at clinically relevant concentrations. This study was designed to evaluate the direct uncoupling effects of the two COMT inhibitors in vitro and in vivo. In isolated rat liver mitochondria, entacapone had no effect on the membrane potential at therapeutical concentrations, but both tolcapone and the reference compound 2,4-dinitrophenol disrupted the potential at low microM concentrations. Since protein binding is speculated to decrease the uncoupling effects in vivo, the COMT inhibitory effect of entacapone and tolcapone as a surrogate for the overall activity of these inhibitors was evaluated in vitro with or without serum. The COMT inhibitory activity of entacapone was reduced to half, while tolcapone had only about 1/10 of its activity left in the presence of serum. Further, uncoupling is known to induce an increase in the body temperature in vivo, and these effects were evaluated in the rat by a possible hyperthermic response to the treatment with entacapone or tolcapone in combination with L-dopa (10 mg/kg) and carbidopa (20 mg/kg). This combination with entacapone (400 mg/kg) had no effect on the rectal body temperature. In contrast, tolcapone (50 mg/kg) caused an elevation in the body temperature together with L-dopa and carbidopa (P < 0.01). Both in vitro and in vivo results indicate that entacapone does not impair energy metabolism related to uncoupling of oxidative phosphorylation.