Tatsuaki Matsubara

Characteristic patterns of circadian variation in plasma catecholamine levels, blood pressure and heart rate variability in Type 2 diabetic patients

Aims To investigate whether Type 2 diabetic patients exhibit characteristic patterns of circadian variation in plasma levels of catecholamines, blood pressure (BP) and heart rate variability (HRV).

Elevated levels of pro-inflammatory cytokines in coronary artery thrombi.

To validate the hypothesis that artery sites occluded with thrombi release pro-inflammatory cytokines, we measured concentrations of interleukin (IL)-6 and IL-8 in infarct-related coronary artery thrombi and atherosclerotic plaque specimens obtained with a transluminal extraction catheter (TEC) from cases of acute myocardial infarction (MI). Fifteen patients (group I) were enrolled in the study and four sets of samples were obtained (taken from the right atrium both before and after angioplasty, from infarct-related coronary artery thrombi and atherosclerotic plaque aspirated with a TEC and from the thoracic aorta aspirated with a TEC). Ten patients undergoing elective TEC served as controls (group II). IL-6 and IL-8 were measured in all patients by means of an enzyme-linked immunosorbent assay. Both IL-6 and IL-8 levels of infarct-related coronary artery samples in group I were significantly higher than in group II (mean +/- SEM, 15.3+/-4.5 vs. 3.8+/-1.2 pg/ml; P<0.01 and 44.0+/-2.4 vs. 15.6+/-0.6 pg/ml; P<0.01, respectively). The results suggest that pro-inflammatory cytokines originate from occluded coronary arteries in acute MI.

Ca2+-dependent Modulation of Intracellular Mg2+ Concentration with Amiloride and KB-R7943 in Pig Carotid Artery

It has long been recognized that magnesium is associated with several important diseases, including diabetes, hypertension, cardiovascular, and cerebrovascular diseases. In the present study, we measured the intracellular free Mg2+ concentration ([Mg2+]i) using 31P nuclear magnetic resonance (NMR) in pig carotid artery smooth muscle. In normal solution, application of amiloride (1 mm) decreased [Mg2+]i by ∼12% after 100 min. Subsequent washout tended to further decrease [Mg2+]i. In contrast, application of amiloride significantly increased [Mg2+]i (by ∼13% after 100 min) under Ca2+-free conditions, where passive Mg2+ influx is facilitated. The treatments had little effect on intracellular ATP and pH (pHi). Essentially the same Ca2+-dependent changes in [Mg2+]i were produced with KB-R7943, a selective blocker of reverse mode Na+-Ca2+ exchange. Application of dimethyl amiloride (0.1 mm) in the presence of Ca2+ did not significantly change [Mg2+]i, although it inhibited Na+-H+ exchange at the same concentration. Removal of extracellular Na+ caused a marginal increase in [Mg2+]i after 100–200 min, as seen in intestinal smooth muscle in which Na+-Mg2+ exchange is known to be the primary mechanism of maintaining a low [Mg2+]i against electrochemical equilibrium. In Na+-free solution (containing Ca2+), neither amiloride nor KB-R7943 decreased [Mg2+]i, but they rather increased it. The results suggest that these inhibitory drugs for Na+-Ca2+ exchange directly modulate Na+-Mg2+ exchange in a Ca2+-dependent manner, and consequently produce the paradoxical decrease in [Mg2+]i in the presence of Ca2+.

Insulin modulation of intracellular free magnesium in heart: involvement of protein kinase C

In the present study of rat heart using 31P‐nuclear magnetic resonance, we examined the interaction between β‐adrenergic and insulin receptors in terms of the intracellular free Mg2+ concentration ([Mg2+]i) regulation. [Mg2+]i was estimated from the separation of the chemical shifts of the α‐ and β‐adenosine triphosphate (ATP) peaks, using the dissociation constant of MgATP 87u2003μM (established recently). In normal (phosphate‐free Krebs‐Henseleit) solution, [Mg2+]i was approximately 1.02u2003mM. Insulin at physiological and pathological concentrations increased [Mg2+]i and contractility in a dose‐dependent manner. Insulin (more than 100u2003μuu2003ml−1) suppressed the decrease in [Mg2+]i caused by isoprenaline (100u2003nM), and these effects of insulin on [Mg2+]i and contractility were blocked by LY333531 (macrocyclic bis (indolyl) maleimide, 100u2003nM), a protein kinase C (PKC) inhibitor. The isoprenaline‐induced decrease in the concentrations of ATP ([ATP]) with insulin application was significantly smaller than that without insulin. Insulin modulates [Mg2+]i and haemodynamics, presumably via activation of PKC, thereby antagonizing the reduction of [Mg2+]i induced by β‐adrenoceptor stimulation.

Stimulation of muscarinic cholinoceptive neurons in the hippocampus evokes a pressor response with bradycardia

The injection of neostigmine into the hippocampus of anesthetized rats increased the mean arterial blood pressure (17% of baseline after 60 min injection) and decreased the heart rate (24% of baseline after 60 min injection). These changes were blocked by the co-administration of methylatropine into the hippocampus. Intrahippocampal injection of neostigmine stimulated the secretion of epinephrine and norepinephrine. Adrenodemedullation did not suppress the increase in blood pressure and the decrease in heart rate. It is concluded that the stimulation of muscarinic cholinoceptive neurons in the hippocampus evokes a hypertensive response via an increase in sympathetic drive to the heart and peripheral vasculature, with bradycardia possibly mediated via the parasympathetic system.

Dynamics of Ca2+/calmodulin-dependent protein kinase II following acute myocardial ischemia—translocation and autophosphorylation

Ca(2+)/calmodulin-dependent protein kinase (CaMK) family is responsive to changes in the intracellular Ca(2+) concentration. However, their functions have not been well established in the ischemia/reperfusion heart. The effects of myocardial ischemia on CaMKII, the most strongly expressed form, were investigated using isolated rat hearts. Rat hearts were rendered globally ischemic by stopping perfusion for 15 min, and then reperfused, heart ventricles being analyzed in each phase. Western blotting detected a decrease in the cytosolic and concomitant increase in the particulate fraction of CaMKII following transient ischemia. Redistribution to the cytosol was revealed on reperfusion. Northern blot showed CaMKII gene expression decreased by ischemia. Furthermore, autoradiography and confocal immunohistochemical findings provided autophosphorylation of CaMKII in the cytosol, ischemia causing decrease, with gradual recovery on reperfusion. These results indicate a transient partial translocation of CaMKII accompanied by kinase activity, with residual myocardial CaMKII undergoing autophosphorylation during ischemia and reperfusion, demonstrating two different characteristic dynamics of CaMKII.

Effects of prolonged application of isoprenaline on intracellular free magnesium concentration in isolated heart of rat

1 The effect of prolonged application of isoprenaline on intracellular free‐Mg2+ concentration ([Mg2+]i) was examined by use of 31P‐nuclear magnetic resonance (31P‐n.m.r.) in rat isolated hearts. Left ventricular pressure (LVP) was simultaneously measured. 2 [Mg2+]i was estimated from the separation of the α‐ and β‐ATP peaks, using the dissociation constant of MgATP 38 μm (established previously). In normal (phosphate‐free, Krebs‐Henseleit) solution, [Mg2+]i was approximately 0.4 mm. 3 When isoprenaline was applied for 100 min, a transient increase in [Mg2+]i was observed during the initial 25 min, whilst concentrations of ATP ([ATP]) and phosphocreatine ([PCr]) decreased and [Pi] correspondingly increased. During the subsequent 75 min of isoprenaline application, [Mg2+]i decreased below its resting levels. Washout of isoprenaline restored [Mg2+]i and [PCr], but [ATP] remained low. These changes elicited by isoprenaline were not observed in the presence of propranolol, a typical α‐adrenoceptor blocker. 4 Isoprenaline increased both LVP and heart rate. The increased LVP and heart rate slowly returned to lower values during prolonged application of isoprenaline, but remained higher than those before application. 5 The transient rise in [Mg2+]i elicited by isoprenaline could be attributed to the decrease in [ATP] resulting in a release of Mg2+. The subsequent decrease in [Mg2+]i during the prolonged applications suggests that β‐adrenoceptor stimulation itself facilitates Mg2+‐extruding mechanism(s).

Relationship between regional myocardial blood flow and mitochondrial function.

SummaryThe purpose of this study was to clarify the relationship between myocardial mitochondrial dysfunction and the degree plus duration of restricted coronary blood flow. 135 anesthetized and open-chest dogs were divided into 3 groups according to coronary occlusion time: 10, 20, and 60 min. Regional myocardial blood flow (MBF) was determined in both ischemic and nonischemic areas before and during coronary occlusion using the hydrogen gas clearance method. Myocardial mitochondria were prepared from each area in which MBF was determined after 10, 20, or 60 min of coronary ligation, and their respiratory control index (RCI), ADP/O, and rate of oxygen consumption in state III O2 (St. III O2) were measured. The MBF measured in 135 dogs before coronary ligation was 103±25 ml/min/100 g (mean±SD) for the area to be rendered ischemic and 101±24 ml/min/100 g for the control area. The MBF in the ischemic area did not cease completely following coronary ligation, and the distribution of MBF showed variations which seemed atributable to individual differences. In the 10-min group, no index of mitochondrial function of the ischemic area differed from that of the nonischemic area at any level of MBF. When MBF was less than 20 ml/min/100 g, RCI of mitochondria from the ischemic area was significantly lower than that from the nonischemic area, in the 20- and 60-min groups. When MBF was less than 20 ml/min/100 g, St. III O2 of mitochondria from the ischemic area significantly decreased compared with that from the nonischemic area, in the 20-min group. In the 60-min group, MBF less than 30 ml/min/100 g, St. III O2 of mitochondria from the ischemic area was likewise significantly decreased. Moreover, with MBF below 20 ml/min/100 g, both RCI and St. III O2 of mitochondria from the ischemic area were significantly lower in the 60-min group than in the 20-min group. These results indicate that ischemia-induced mitochondrial dysfunction depends on the degree of decrease in the blood flow of the area involved as well as on the duration of ischemia, and the blood flow that is critical for survival, based on mitochondrial function, is approximately 20 ml/min/100 g, i.e., a reduction to 20% of normal value.

Pressor response induced by the hippocampal administration of neostigmine is suppressed by M1 muscarinic antagonist

We investigated the roles played by three muscarinic receptors (M1, M2, and M3) in the pressor response with bradycardia that followed the injection of neostigmine (5 x 10(-8) mol) into the hippocampus of anesthetized rats. These changes were blocked by the co-administration of methylatropine (5 x 10(-8) mol). The intrahippocampal injection of pirenzepine (M1 antagonist) (5 x 10(-9) - 5 x 10(-7) mol) suppressed the neostigmine-induced pressor response dose-dependently. However injection of gallamine (M2 antagonist) (5 x 10(-8) - 5 x 10(-7) mol) and of 4-DAMP (M1 and M3 antagonist) (5 x 10(-8) - 5 x 10(-7) mol) did not suppress this hypertensive response. These findings suggest that the neostigmine-induced pressor response with bradycardia is mediated through the M1 muscarinic receptor subtype.

Effects of diabetes on cardiac glycogen metabolism in rats

SummaryThe effects of diabetes on myocardial glycogen metabolism in rats were examined and compared with those of fasting. Male Wistar rats were divided into three groups: controls, streptozotocininduced diabetics, and one-week fasted. Isolated rat hearts were subjected to substrate-free 30-min Langendorff perfusion followed by 60-min working heart perfusion with glucose alone or in combination with insulin or insulin plus β-hydroxybutyrate (BHB). Myocardial glycogen contents were determined before or 30 min after Langendorff perfusion, or 60 min after working heart perfusion. Before Langendorff perfusion, tissue glycogen concentrations in control, diabetic, and fasted hearts were 3.3 ± 0.2, 10.0 ± 0.9, and 5.7 ± 0.5 (mg/g wet weight), respectively. In diabetic rats, the myocardial glycogen concentration was markedly decreased after working heart perfusion of any of the substrate combinations, even those with insulin and BHB. In contrast, myocardial glycogen in control or fasted rats was not reduced after the addition of glucose with insulin, and/or glucose with insulin and BHB. These results suggest that degradation of tissue glycogen occurs in isolated perfused hearts from diabetic rats, while a clearly different response is shown by fasted hearts.