Potent Cardioprotective Effect of the 4-Anilinoquinazoline Derivative PD153035: Involvement of Mitochondrial KATP Channel Activation

Background: The aim of the present study was to evaluate the protective effects of the 4-anilinoquinazoline derivative PD153035 on cardiac ischemia/reperfusion and mitochondrial function. Methodology/Principal Findings: Perfused rat hearts and cardiac HL-1 cells were used to determine cardioprotective effects of PD153035. Isolated rat heart mitochondria were studied to uncover mechanisms of cardioprotection. Nanomolar doses of PD153035 strongly protect against heart and cardiomyocyte damage induced by ischemia/reperfusion and cyanide/aglycemia. PD153035 did not alter oxidative phosphorylation, nor directly prevent Ca(2+) induced mitochondrial membrane permeability transition. The protective effect of PD153035 on HL-1 cells was also independent of AKT phosphorylation state. Interestingly, PD153035 activated K(+) transport in isolated mitochondria, in a manner prevented by ATP and 5-hydroxydecanoate, inhibitors of mitochondrial ATP-sensitive K(+) channels (mitoK(ATP)). 5-Hydroxydecanoate also inhibited the cardioprotective effect of PD153035 in cardiac HL-1 cells, demonstrating that this protection is dependent on mitoK(ATP) activation. Conclusions/Significance: We conclude that PD153035 is a potent cardioprotective compound and acts in a mechanism involving mitoK(ATP) activation

Long-term intermittent feeding, but not caloric restriction, leads to redox imbalance, insulin receptor nitration, and glucose intolerance

Calorie restriction is a dietary intervention known to improve redox state, glucose tolerance, and animal life span. Other interventions have been adopted as study models for caloric restriction, including nonsupplemented food restriction and intermittent, every-other-day feedings. We compared the short- and long-term effects of these interventions to ad libitum protocols and found that, although all restricted diets decrease body weight, intermittent feeding did not decrease intra-abdominal adiposity. Short-term calorie restriction and intermittent feeding presented similar results relative to glucose tolerance. Surprisingly, long-term intermittent feeding promoted glucose intolerance, without a loss in insulin receptor phosphorylation. Intermittent feeding substantially increased insulin receptor nitration in both intra-abdominal adipose tissue and muscle, a modification associated with receptor inactivation. All restricted diets enhanced nitric oxide synthase levels in the insulin-responsive adipose tissue and skeletal muscle. However, whereas calorie restriction improved tissue redox state, food restriction and intermittent feedings did not. In fact, long-term intermittent feeding resulted in largely enhanced tissue release of oxidants. Overall, our results show that restricted diets are significantly different in their effects on glucose tolerance and redox state when adopted long-term. Furthermore, we show that intermittent feeding can lead to oxidative insulin receptor inactivation and glucose intolerance. (C) 2011 Elsevier Inc. All rights reserved.FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao PauloFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Instituto Nacional de Ciencia e Tecnologia de Processos Redox em Biomedicina (INCT)Instituto Nacional de Ciencia e Tecnologia de Processos Redox em Biomedicina (INCT)CNPq Conselho Nacional de Desenvolvimento Cientifico e TecnologicoConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CAPES Coordenacao de Aperfeicoamento do Pessoal de Nivel SuperiorCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)John Simon Guggenheim Memorial FoundationJohn Simon Guggenheim Memorial Foundatio

PD153035 does not alter AKT phosphorylation.

<p>Cardiac HL-1 cells were preincubated in standard media (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010666#s2" target="_blank">Materials and Methods</a>) containing 10 nM PD153035 or the equivalent concentration of PD153035 solvent DMSO during 20 min. 10 mM K-cyanide (CN) and 2 mM 2-deoxyglucose (2-DG) were then added under the conditions of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010666#pone-0010666-g002" target="_blank">Figure 2</a>, where indicated. After 60 min, suspension proteins were extracted and AKT and pAKT levels were estimated by immunoblotting. *p<0.01 relative to the conditions in the absence of “CN+2-DG”.</p

PD153035 does not decrease mitochondrial Ca²⁺ uptake, nor prevent permeability transition.

<p>Isolated rat heart mitochondria (RHM; 0.5 mg/mL) were incubated in 125 mM sucrose, 65 mM KCl, 10 mM HEPES, 2.5 mM KH₂PO₄, 40 µM arsenazo III, 1.25 mM malate, 1.25 mM glutamate, 1.25 mM pyruvate and 1.25 mM α-ketoglutarate, pH 7.2 (KOH). Ca²⁺ was added where indicated in four consecutive boluses of 10 µM, totaling 40 µM. <i>Line a</i> represents a control experiment with no further additions. <i>Line b</i> represents an experiment conducted in the presence of 1 µM cyclosporin A. <i>Lines c–f</i> represent experiments conducted in the presence of 1 nM, 10 nM, 30 nM and 100 nM PD153035, sequentially. Data are representative traces of 3 similar repetitions.</p

PD153035 does not change mitochondrial respiratory parameters.

<p>Rat heart mitochondria were incubated in 125 mM sucrose, 65 mM KCl, 10 mM HEPES, 2.5 mM KH₂PO₄, 0.4 mM EGTA, 1.25 mM malate, 1.25 mM glutamate, 1.25 mM pyruvate and 1.25 mM α-ketoglutarate, pH 7.2 (KOH), in the presence of 10 nM PD153035 or the equivalent concentration of PD153035 solvent DMSO (0.1%) (Control), as indicated. ADP (250 µM) and 1 µg/mL oligomycin were added to achieve state 3 and 4 respiratory rates, respectively. Respiration was measured using a Clark-type electrode, and respiratory parameters were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010666#s2" target="_blank">Materials and Methods</a>. None of the values obtained in the presence of PD153035 are significantly different from controls (four independent preparations, experiments performed in triplicate).</p