Toshiki Iwata

Bradykinin B2 Receptor Gene Polymorphism Is Associated With Angiotensin-Converting Enzyme Inhibitor–Related Cough

The appearance of cough in association with angiotensin-converting enzyme (ACE) inhibitors is thought to be related to bradykinin, and it has been speculated that the elicitation of adverse effects is genetically predetermined. Several polymorphisms of the human bradykinin B(2) receptor gene may be involved in ACE inhibitor-related cough. To investigate this possibility, we identified the -58 thymine (T)/cytosine (C) polymorphism in subjects with ACE inhibitor-related cough. We classified the study population into 4 groups: subjects with and without cough that were treated with ACE inhibitors (n=30/30), nontreated essential hypertensive subjects (n=100), and normotensive subjects (n=100). The -58T/C was genotyped by the polymerase chain reaction single-strand conformation polymorphism method. The frequencies of the CC genotype and C allele of -58T/C were significantly higher in the nontreated hypertensive subjects than in the normotensive subjects. Conversely, the frequencies of the TT genotype and T allele were significantly higher in the subjects with cough than in the subjects without cough. These tendencies were more pronounced in females. Among the promoter assays of the human bradykinin B(2) receptor, -58T was found to have a higher transcription rate than that of -58C. This finding seems to suggest that the transcriptional activity of promoter might be involved in the appearance of ACE inhibitor-related cough. A genetic variant of the bradykinin receptor is involved in the elicitation of ACE inhibitor-related cough. It may be possible to predict the side effects of ACE inhibitors in advance.

Histochemical studies of myocardial injury caused by hydrogen peroxide after reperfusion using the cerium method

Free radicals have been implicated in myocardial reperfusion injury. Hydrogen peroxide (H2O2) is a precursor of highly reactive oxygen intermediates. In this study, we investigated myocardial injury caused by endogenous H2O2 during the early reperfusion period following brief ischemia with electron microscopy and the cerium method. This method involves formation of an electrondense precipitate when H2O2 reacts with cerium chloride (CeCl3). We used isolated, functioning hearts prepared according to the working heart model, which were reperfused with a solution containing 0.5mM CeCl3 for 5 min after 10 min of ischemia. Some hearts were treated with 3-amino-1,2,4-triazole (ATZ) to inhibit catalase; others were treated with ATZ and superoxide dismutase (SOD), which dismutates the superoxide anion to hydrogen peroxide. In the control group (no drugs given) and the ATZ-treated group, the CeCl3−H2O2-dependent reaction products during the reperfusion period appeared in 12% and 28%, respectively, of the microvascular spaces. Treatment with SOD did not produce a decrease in electron-dense precipitates or a decrease in myocardial injury during ischemia-reperfusion. Moreover, in the ATZ group, moderately injured myocytes were seen (swelling of mitocondria, intermyofibrillar edema). Our results indicate that in myocytes, catalase plays an important role in the defense against H2O2 and that the increase in H2O2 is a cause of reperfusion injury. However, SOD does not protect against H2O2 in the absence of catalase.

Oxygen Consumption and Mitochondrial Membrane Potential in Postischemic Myocardium

Oxygen consumption may be disproportionately high relative to contractile function in postischemic reperfused myocardium. The study reported in this chapter investigated the mechanism of the dissociation between oxygen consumption and contractile function in postischemic reperfused myocardium using isolated rat hearts. Mitochondrial dysfunction secondary to increased calcium uptake has been implicated as an important mediator of reperfusion injury in the heart. In postischemic, isovolumic, antegrate-perfused rat hearts, the myocardial oxygen consumption rate (MVO2) and contractile function were studied in relation to mitochondrial function. Left ventricular pressure, coronary blood flow, and oxygen consumption were determined. Mitochondrial respiration and the mitochondrial membrane potential were measured by polarography and flow cytometry, respectively. To examine the role of mitochondrial calcium uptake in ischemia reperfusion injury, isolated rat hearts perfused with ruthenium red, which inhibits calcium uptake by mitochondria, were compared to control perfused hearts. After stabilization, hearts were subjected to 60 minutes of no-flow ischemia, followed by 60 minutes of reperfusion. At 15 minutes after the onset of reperfusion, there was poor recovery of left ventricular developed pressure to 64% of the control level, but myocardial oxygen consumption was increased to 134% of control. The addition of 2.5 μM ruthenium red to the perfusate resulted in a decrease of myocardial oxygen consumption. The oxygen consumption rate in state 3 of mitochondria decreased similarly following reperfusion in control and ruthenium red hearts. The mitochondrial membrane potential was reduced to 89% (logarithmic scale) after 15 minutes of reperfusion and then returned to preischemic level. These data suggest that the dissociation between oxygen consumption and contractile function following early reperfusion is partly caused by the repair of intracellular damage resulting from calcium accumulation to mitochondria.

Production of Hydrogen Peroxide During Hypoxia-Reoxygenation in Isolated Myocytes

Active oxygen species, including hydrogen peroxide (H2O2), have been implicated in myocardial reperfusion injury. Recently, spin-trap agents and biochemical techniques applied to intact hearts have shown that H2O2 is generated by leukocytes, by endothelial cells, and by mitochondria in myocytes. In this study, we used electron microscopy and the cerium (Ce) method to histologically investigate H2O2 formation during hypoxia—reoxygenation and its toxic effects on myocardium. This Ce method involves the formation of an electron-dense precipitate when H2O2 reacts with cerium chloride (CeCl3). Single myocytes were obtained from rat hearts by the collagenase method. Isolated myocytes were reoxygenated for 15 minutes after 30 minutes of hypoxia. Digitonin and CeCl3, were added to make cell membranes permeable and to detect intracellular H2O2 by electron microscopy. In the control group, the ultrastructure was well preserved and no dense deposits were found in myocytes. However, in the hypoxia—reoxygenation group, precipitates, which were cerium—H2O2 reaction products, were found along swollen mitochondria. Moreover, in the hypoxia-reoxygenation group, cell viability was reduced to 72% of control. These results indicate that H2O2 is generated by mitochondria and that its relese into cytosol may lead to myocyte death during hypoxia—reperfusion.

Effects of Catecholamine and Amrinone on the Metabolism of Noninfarcted Myocardium in Cardiogenic Shock

We investigated the effects of catecholamine and amrinone (AMR) on the metabolism of noninfarcted myocardium (NIM) during heart failure in acute myocardial infarction. Acute myocardial ischemia was induced by left circumflex coronary artery ligation on dogs divided into two groups: in the C group, left ventricular pressive (LVP) remained at >70% and in the S group, LVP decreased to <70% of preligation values. In part of the S group, 10 μg/kg/min of dopamine (DOA) or dobutamine (DOB), or 60μg/kg/min of AMR, were given intravenously beginning 90 min after ligation. At the end of 120 min of ischemia, mitochondria were extracted from NIM, and respiratory and electron transport system enzyme activities were measured. In the DOA and DOB groups, LVP, myocardial blood flow, cardiac output, and max LV dp/dt recovered significantly. In the AMR group, in spite of LVP reduction, other hemodynamic parameters increased. In the S group, state III respiration, complex I, and DNP-ATPase activities in NIM decreased to 62%, 65%, and 68% of preligaton levels, respectively. These values improved markedly with DOA, DOB, and AMR treatments. Electron microscopy showed swelling and fusion of mitochondria in the S group. These results indicate that catecholamine and AMR improve energy production in NIM and ultimately improve cardiac function.

Metabolic Changes in Nonischemic Myocardium During Pump Failure

Metabolic changes in the nonischemic myocardium after acute myocardial infarction in canine hearts were studied. Ca2+-ATPase activity and the major ATPase protein of the sarcoplasmic reticulum, tissue levels of ATP, mitochondrial respiratory, and complex I activities were decreased in the noninfarcted zone in proportion to heart function. It is suggested that recovery of these functions may be important in any treatment of pump failure.