Norio Shimamoto

Contribution of endogenous endothelin to the extension of myocardial infarct size in rats.

Pathophysiological roles of endogenous endothelin have been studied from the viewpoint of its contribution to the extension of myocardial infarct size. A monoclonal antibody against endothelin 1 (AwETN40) suppressed changes induced by endothelin 1 and endothelin 2 but did not modify those by endothelin 3 in vivo or in vitro. Effects of AwETN40 on myocardial infarct size were investigated. Coronary ligation (1 hour) and reperfusion (24 hours) in rats caused infarction in 35% of the left ventricle. Repetitive or single administration of AwETN40 reduced the infarct size; an intravenous injection of 22.5 mg/kg of the antibody 5 minutes after coronary occlusion or 5 minutes before reperfusion reduced the size by 38% or 31% of the control, respectively. Plasma and tissue endothelin 1 and plasma big endothelin 1 in rats were measured at various stages after occlusion. Plasma endothelin 1 showed a fourfold increase 10 minutes after reperfusion (from 1.02 to 3.96 pg/ml) and had returned to the control value after 8 hours. Plasma big endothelin 1 showed changes similar to those of plasma endothelin 1. No significant changes in plasma endothelin 2 and endothelin 3 were observed. Cardiac tissue contained seven times as much endothelin 1 as the control value 1 hour after reperfusion (4.59 versus 33.1 pg/g tissue), and a high concentration (13.2 pg/g tissue) was maintained even after 48 hours. We concluded that an increase in endogenous endothelin 1 plays an important role in the extension of myocardial infarct size.

Contribution of Sustained Ca Elevation for Nitric Oxide Production in Endothelial Cells and Subsequent Modulation of Ca Transient in Vascular Smooth Muscle Cells in Coculture

To elucidate the intracellular Ca (Ca) transient responsible for nitric oxide (NO) production in endothelial cells (ECs) and the subsequent Ca reduction in vascular smooth muscle cells (VSMCs), we administrated four agonists with different Ca-mobilizing mechanisms for both cells in iso- or coculture. We monitored the Ca of both cells by two-dimensional fura-2 imaging, simultaneously measuring NO production as NO. The order of potency of the agonists in terms of the peak Ca in ECs was bradykinin (100 nM) > ATP (10 μM) > ionomycin (50 nM) > thapsigargin (1 μM). In contrast, the order in reference to both the extent of Ca reduction in cocultured VSMCs and the elevation in NO production over the level of basal release in ECs completely matched and was ranked as thapsigargin > ionomycin > ATP > bradykinin. Treatment by N-monomethyl-L-arginine monoacetate but not indomethacin or glybenclamide restored the Ca response in cocultured VSMCs to the isoculture level. In ECs, when the Ca influx was blocked by Ni or by chelating extracellular Ca, all four agonists markedly decreased NO production, the half decay time of the Ca degenerating phase, and the area under the Ca curve. The amount of produced NO hyperbolically correlated to the half decay time and the area under the Ca curve but not to the Ca peak level. Thus, the sustained elevation of Ca in ECs, mainly a result of Ca influx, determines the active NO production and subsequent Ca reduction in adjacent VSMCs. Furthermore, L-arginine but not D-arginine or L-lysine at high dose (5 mM) without agonist enhanced the NO production, weakly reduced the Ca in ECs, and markedly decreased the Ca in VSMCs, demonstrating the autocrine and paracrine effects of NO (Shin, W. S., Sasaki, T., Kato, M., Hara, K., Seko, A., Yang, W. D., Shimamoto, N., Sugimoto, T., and Toyo-oka, T.(1992) J. Biol. Chem. 267, 20377-20382).

Positive Inotropic and Vasoconstrictive Effects of Endothelin-1 in in vivo and in vitro Experiments: Characteristics and the Role of L-Type Calcium Channels

Summary Cardiac and vascular effects of endothelin-1 (ET-1) were studied in hemodynamic models in vivo and in isolated artery and heart tissues. The hemodynamic changes induced by ET-1 (30–300 pmol/kg i.v.) in dogs were increases in blood pressure, cardiac output, and ventricular contractility. Ring preparations of canine coronary artery and rabbit thoracic aorta showed vasoconstrictive responses to ET-1 (10–10 M and over) with an EC50 of 3–10

Ontogeny of pituitary adenylate cyclase-activating polypeptide (PACAP) and its binding sites in the rat brain

10–9 M. Nifedipine 10–5 M did not reverse these effects completely. ET-1 had positive inotropic effects from 10–10 M in rabbit papillary muscles. It lengthened the action potential duration by 16% and increased the developed tension by 180% (10–8 M). In contrast, Bay K 8644 (10–7 M) produced similar lengthening (17%) in the duration with only a 37% increase in the tension. This suggests a difference in the mechanisms of the positive inotropic effects between these two agents. We conclude that ET-1 has positive inotropic and vasoconstrictive effects, and that these ET-1-induced contractions in ventricular muscles and arteries cannot be explained thoroughly by the increase in currents through voltage-dependent calcium channels.

Myocardial energy metabolism in the hypertrophied hearts of spontaneously hypertensive rats

We have previously shown that inhibition of catalase and glutathione peroxidase activities by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS), respectively, in rat primary hepatocytes caused sustained endogenous oxidative stress and apoptotic cell death without caspase-3 activation. In this study, we investigated the mechanism of this apoptotic cell death in terms of nucleosomal DNA fragmentation. Treatment with ATZ+MS time-dependently increased the number of deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)-positive nuclei from 12 h, resulting in clear DNA laddering at 24 h. The deoxyribonuclease (DNase) inhibitor, aurintricarboxylic acid (ATA), completely inhibited nucleosomal DNA fragmentation but the pan-caspase inhibitor, z-VAD-fmk was without effects; furthermore, the cleavage of inhibitor of caspase-activated DNase was not detected, indicating the involvement of DNase(s) other than caspase-activated DNase. Considering that endonuclease G (EndoG) reportedly acts in a caspase-independent manner, we cloned rat EndoG cDNA for the first time. Recombinant EndoG alone digested plasmid DNA and induced nucleosomal DNA fragmentation in isolated hepatocyte nuclei. Recombinant EndoG activity was inhibited by ATA but not by hydrogen peroxide, even at 10 mm. ATZ+MS stimulation elicited decreases in mitochondrial membrane potential and EndoG translocation from mitochondria to nuclei. By applying RNA interference, the mRNA levels of EndoG were almost completely suppressed and the amount of EndoG protein was decreased to approximately half the level of untreated cells. Under these conditions, decreases in TUNEL-positive nuclei were significantly suppressed. These results indicate that EndoG is responsible, at least in part, for nucleosomal DNA fragmentation under endogenous oxidative stress conditions induced by ATZ+MS.

Cardiovascular effects of endothelin in dogs: positive inotropic action in vivo

Endogenous levels of pituitary adenylate cyclase-activating polypeptide (PACAP) and its binding site densities were measured in eight brain regions in rats of different ages (2-240 days) by sandwich-enzyme immunoassay and autoradiography. PACAP levels were quite low at day 2, peaked in 30-60 days, and then remained constant in most regions. Such ontogenetic changes are similar to those of vasoactive intestinal polypeptide (VIP) and classical neurotransmitters. PACAP-binding sites were already dense at day 2 and varied only slightly up to day 240. These results suggest that PACAP may have modulatory effects on brain development.

Critical role of exposure time to endogenous oxidative stress in hepatocyte apoptosis

SummaryAge-related changes in the myocardial energy metabolism were studied in spontaneously hypertensive (SHR) rats of 5–25 weeks of age. Systolic blood pressure increased rapidly during 5 to 10 weeks of age (developing phase) and attained a plateau level at 10 to 15 weeks (sustained phase). Even during the developing phase, the heart was hypertrophic, as assessed by an increase in the ratio of the ventricular weight to body weight. However, myocardial contents of glycolytic intermediates and high energy phosphate compounds and thus, the myocardial energy state (phosphorylation potential) in SHR rats did not differ from those in age-matched normotensive Wistar-Kyoto (WKY) rats. The lactate/pyruvate ratio was significantly lower in SHR rats. On the other hand, during the sustained phase, cardiac hypertrophy progressed only gradually, and myocardial contents of creatine phosphate and ATP were lower, while the lactate content was higher than in WKY rats. The lactate/pyruvate ratio was elevated, while phosphorylation potential was lowered. These findings suggest that the energy state is normal during the developing phase of hypertension despite the presence of cardiac hypertrophy and the increased pressure load, whereas the energy state is at a lower level during the sustained phase of hypertension.ZusammenfassungDie altersabhängigen Änderungen des myokardialen Energieumsatzes wurden bei spontanhypertensiven, 5 bis 25 Wochen alten Ratten (SHR) untersucht. Der systolische Blutdruck stieg zwischen der 5. und 10. Lebenswoche rasch an (Entwicklungsphase) und erreichte nach 10 bis 15 Wochen ein Plateau (Dauerphase). Auch während der Entwicklungsphase war das Herz hypertrophiert, gemessen an einem Anstieg des Verhältnisses Ventrikelgewicht: Körpergewicht. Jedoch unterschied sich der Gehalt des Myokards an Intermediärprodukten der Glykolyse und energiereichen Phosphaten und damit bezüglich des energetischen Status (Phosphorylierungspotential) bei SHR-Ratten nicht von gleichaltrigen normotensiven Wistar-Kyoto-Ratten (WKY). Das Lactat-Pyruvat-Verhältnis war bei SHR-Ratten signifikant vermindert. Andererseits nahm die Herzhypertrophie während der Dauerphase nur allmählich zu; der Gehalt an Kreatinphosphat und ATP war geringer, während der Lactatgehalt höher war als bei WKY-Ratten. Das Lactat-Pyruvat-Verhältnis war gesteigert, während das Phosphorylierungspotential vermindert war. Diese Befunde lassen vermuten, daß der energetische Status während der Entwicklungsphase der Hypertension normal ist trotz des Vorliegens einer Herzhypertrophie und gesteigerter Druckbelastung, während der energetische Status während der Dauerphase herabgesetzt ist.

Effects of pretreatment with 2-O-octadecylascorbic acid, a novel free radical scavenger, on reperfusion-induced arrhythmias in isolated perfused rat hearts.

Endothelin, administered i.v. to anesthetized dogs, dose dependently increased the cardiac output, left ventricular systolic pressure (LVSP), and maximum upstroke velocity (max dp/dt) of the LVSP for about 10 min without changing the heart rate. Thereafter the cardiac output decreased to below the control level but max dp/dt decreased to the control level. The arterial pressure and total peripheral resistance showed an initial, transient decrease followed by a sustained increase. These results suggest that endothelin has positive inotropic and long-lasting vasoconstrictive effects preceded by transient vasodilatation in vivo.

Pharmacologic Profile of EndothelinA/B Antagonist, [Thr18,γMethylLeu19]Endothelin-1

Abstract We have previously shown that inhibition of catalase and glutathione peroxidase activities in rat primary hepatocytes by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS) results in sustained oxidative stress, followed by apoptosis. To examine the effects of duration of oxidative stress, ATZ and MS were removed from culture medium at 3, 6 and 9 h after treatment with both inhibitors. Oxidative stress was induced for periods of time by ATZ and MS exposures in primary hepatocytes. Treatment with ATZ and MS reduced catalase (CAT) and glutathione peroxidase (GPx) activities, and decreased CAT and GPx activities recovered to normal values upon withdrawal. Although oxidative stress of up to 6 h duration did not cause cell death, sustained oxidative stress (over 9 h) induced apoptosis. The increase in the glutathione disulfide/reduced glutathione ratio under oxidative stress up to 6 h was transient and reversible, while that due to sustained oxidative stress was irreversible. These results suggest that irreversible redox shifts resulting from sustained oxidative stress play a critical role in the induction of hepatocyte apoptosis in this experimental system.