Yoshiki Yui

Three distinct subpopulations of GABAergic neurons in rat frontal agranular cortex

GABAergic interneurons in the rat frontal cortex were subdivided on the basis of immunoreactivity for calcium binding proteins, neuropeptides and nitric oxide synthase, using double immunofluorescence and mirror image immunohistochemical methods. The results indicate that in this region of the neocortex there are at least three distinct subpopulations of local circuit neurons. The first subgroup consists of parvalbumin-immunoreactive cells. Those do not contain neuropeptide, calretinin or nitric oxide synthase immunoreactivity. A substantial number of parvalbumin-immunoreactive cells in layer II/III were also immunoreactive for calbindin D28k. The second subgroup consists of cells immunoreactive for calretinin. Most were usually immunoreactive for vasoactive intestinal polypeptide as well, but a few cells in layer II/III were immunoreactive for one or the other only. Calretinin-immunoreactive cells do not colocalize parvalbumin, somatostatin or nitric oxide synthase, and only a few colocalize calbindin D28k. The third subgroup consists of cells most of which contain somatostatin, and is entirely separate from the parvalbumin- and calretinin-immunoreactive populations. There was substantial colocalization of somatostatin and calbindin D28k and of somatostatin and neuropeptide Y. Some somatostatin-immunoreactive cells showed nitric oxide synthase immunoreactivity. All of the populations of immunoreactive cells examined in the present study also showed GABA immunoreactivity. About 10% of calbindin D28k-immunoreactive cells and all of those strongly stained for calbindin D28k in layer II/III showed GABA immunoreactivity. Most calbindin D28k-positive cells in deep layers also showed GABA immunoreactivity. These results support that almost all calbindin D28k-immunoreactive non-pyramidal cells are probably GABAergic.

Anti-anginal effect of fasudil, a Rho-kinase inhibitor, in patients with stable effort angina : a multicenter study

Rho-kinase plays an important role in calcium sensitization for vascular smooth muscle (VSMC) contraction and may be involved in the inappropriate coronary vasoconstriction during exercise-induced myocardial ischemia. In this multicenter phase II study, the anti-anginal effect of fasudil, which is metabolized to a specific Rho-kinase inhibitor hydroxyfasudil after oral administration, was examined in patients with stable effort angina. In the phase IIa trial, after a 2-week washout period of anti-anginal drugs, 45 patients received increasing doses of fasudil (5, 10, and 20 mg TID for every 2 weeks). The fasudil treatment significantly prolonged the maximum exercise time and the time to the onset of 1-mm ST segment depression on treadmill exercise test (both p < 0.01), whereas blood pressure and heart rate during exercise were unchanged before and after the treatment. Higher doses of fasudil (20 and 40 mg TID) were subsequently tested in 22 patients in the same manner with similar positive results. In the phase IIb trial, after a 2-week washout period of anti-anginal drugs, 125 patients were assigned, in a double-blind manner, to a 4-week oral treatment with a different dose of fasudil (5, 10, 20, or 40 mg TID) and treadmill exercise test was performed before and after the treatment. Again, both maximum exercise time and time to the onset of 1-mm ST segment depression were prolonged in all groups. A significant dose-response relation was noted across the treatment groups for the exercise tolerance index that was determined by the combined effect of exercise time and ST segment depression (p = 0.006). Fasudil was well tolerated in both trials without any serious adverse reactions. These results suggest the efficacy and adequate safety profile of fasudil, the first drug in a novel class of vasodilators, for the treatment of stable effort angina.

Prostacyclin therapy in patients with congestive heart failure

The acute hemodynamic effects of intravenous prostacyclin (PGI2), in doses of 22 +/- 11 ng/kg per min were studied in nine patients with severe congestive heart failure refractory to digitalis and diuretic drugs. After prostacyclin infusion, mean (+/- standard deviation) pulmonary capillary wedge pressure decreased from 21.0 +/- 7.9 to 15.0 +/- 6.6 mm Hg (p less than 0.001), mean arterial pressure from 98.9 +/- 12.8 to 76.2 +/- 7.0 mm Hg (p less than 0.001), systemic vascular resistance from 2,574 +/- 384 to 1,368 +/- 283 dynes s cm-5 (p less than 0.001), pulmonary vascular resistance from 1,008 +/- 451 to 443 +/- 135 dynes s cm-5 (p less than 0.001) and pulmonary arteriolar resistance from 330 +/- 111 to 189 +/- 73 dynes s cm-5 (p less than 0.001). Heart rate increased from 78 +/- 21 to 82 +/- 24 beats/min (p = not significant [NS]), cardiac index from 2.0 +/- 0.37 to 3.2 +/- 0.59 liters/min per m2 (p less than 0.001) and stroke index from 27.6 +/- 8.69 to 42.0 +/- 0.62 cc/m2 (p less than 0.001). With prostacyclin, moreover, coldness of the limbs and face disappeared, and patients felt warmth and mild flushing of the face. After prostacyclin, plasma norepinephrine levels, renin activity and aldosterone concentrations rose from 824 +/- 375 to 880 +/- 468 pg/ml (NS), 0.68 +/- 1.36 to 0.95 +/- 1.21 ng/ml per h (NS), and 6.64 +/- 2.50 to 6.38 +/- 2.88 ng/dl (NS), respectively, while plasma epinephrine increased from 140 +/- 80 to 250 +/- 154 pg/ml (p less than 0.025).

Effects of superoxide dismutase on reperfusion arrhythmias and left ventricular function in patients undergoing thrombolysis for anterior wall acute myocardial infarction

Abstract Oxygen-derived free radicals have been implicated as important factors involved in the genesis of reperfusion injury. There is increasing evidence that a free radical scavenger, superoxide dismutase (SOD), limits reperfusion injury in animal experiments.1,2 We administered human recombinant SOD to patients with anterior wall acute myocardial infarction (AMI) at the time of thrombolysis and investigated (1) if SOD can reduce reperfusion arrhythmias, and (2) if SOD has beneficial effects on left ventricular function in the chronic phase.

Immunohistochemical expression of inducible nitric oxide synthase (iNOS) in reversible endotoxic shock studied by a novel monoclonal antibody against rat iNOS.

An antirat monoclonal antibody (mAb) against inducible nitric oxide synthase (iNOS), ANOS11, was used for immunohistochemistry to examine the expression of iNOS in various organs and tissues of adult rats in experimental endotoxic shock induced by lipopolysaccharide (LPS) injection. The phenotype of iNOS‐expressed cells was also examined immunohistochemically using various mAbs. In control rats, very few cells were positive for ANOS11 except in the thymus. After intravenous injection of LPS, the number of iNOS‐positive cells increased rapidly in almost all organs, except the thymus and brain, peaked 6 h after the injection, and decreased slowly. Of the numerous inflammatory cells that infiltrated the lungs, liver, and spleen after LPS injection, many were positive for ANOS11. Besides inflammatory cells, hepatocytes and endothelial cells of the aorta were also positive for ANOS11 but only around 6 h after injection. The cellular composition of iNOS‐positive infiltrated cells changed along with the progression of endotoxic shock. At 4 to 6 h after injection, most iNOS‐positive cells were considered polymorphonuclear leukocytes judging by their positive reactivity to 0X42 and their nuclear morphology. The population of iNOS‐positive macrophages positive for EDI or ED2 increased with time. After 24 h, many iNOS‐positive macrophages were found around the focal necrosis in the liver and spleen. These results indicate that the expression of iNOS in neutrophils, endothelial cells, and hepatocytes precedes that of macrophages in experimental endotoxic shock. The expression of iNOS in various cells and organs is closely associated with the progress and pathological changes of endotoxic shock. J. Leukoc. Biol. 57: 36–44; 1995.

Prostaglandin I2 half-life regulated by high density lipoprotein is decreased in acute myocardial infarction and unstable angina pectoris.

To investigate the prostaglandin I2 (PGI2) half-life regulated by high density lipoprotein (HDL) in patients with coronary artery disease (CAD), we determined the stability of PGI2 and serum apolipoprotein A-I (Apo A-I) and apolipoprotein A-II (Apo A-II) levels in four age-matched groups of patients: controls (n = 17), angina pectoris (n = 18), unstable angina pectoris (n = 17), myocardial infarction (n = 19) (acute phase, 3.6 +/- 1.7 hours from onset; subacute phase, 75 +/- 15 hours from onset in the same patients). Serum PGI2 half-life and total serum Apo A-I levels were lower in the CAD group than in the control group. PGI2 was least stable in patients with unstable angina and the acute phase of myocardial infarction. In these patients, the molar ratio of Apo A-I to Apo A-II and HDL-associated Apo A-I levels were decreased, and free Apo A-I levels were increased. After in vitro incubation of HDL with increasing amounts of Apo A-II, Apo A-I in HDL was displaced by Apo A-II, with the parallel decrease in stability of PGI2. Free Apo A-I cannot stabilize PGI2. HDL-associated Apo A-I, whose amount is affected by Apo A-II, stabilized PGI2 and correlated well with stability of PGI2 in patients with CAD and control patients. Decreased PGI2 half-life may play an important role in the pathogenesis of atherosclerosis and thrombus formation in the coronary arteries, especially thrombus formation during an acute coronary event.

Effects of coronary artery reperfusion on relation between creatine kinase-MB release and infarct size estimated by myocardial emission tomography with thallium-201 in man.

The quantitative relations between serum creatine kinase-MB isoenzyme (CK-MB) release and the final infarct size estimated by myocardial emission computed tomography with thallium-201 was assessed in 37 patients with a first acute transmural myocardial infarction who underwent intracoronary thrombolysis using urokinase 4.6 +/- 1.9 hours after the onset of symptoms. Serial CK-MB determinations were used to calculate the accumulated release of CK-MB (sigma CK-MB). Myocardial emission tomography with thallium-201 was performed 4 weeks after the onset, and infarct volume was measured from reconstructed tomographic images by computerized planimetry. The results are presented for two groups of patients: 11 patients with unsuccessful thrombolysis (group A) and 26 patients with successful thrombolysis (group B). An excellent linear relation was found for group A (sigma CK-MB = 6.4 X infarct volume + 47.7, r = 0.91), whereas a different linear relation was observed for group B (sigma CK-MB = 10.5 X infarct volume + 89.1, r = 0.80). Moreover, serum CK-MB activity reached a peak at 21.1 +/- 2.2 hours after the onset in group A and reached an earlier peak at 12.5 +/- 2.9 hours in group B (p less than 0.001). These data suggest that acute coronary recanalization alters the kinetics of CK-MB release, resulting in greater CK-MB release into the serum for equivalent infarct volume estimated by myocardial emission tomography with thallium-201. Thus, serum CK-MB time-activity curves after acute myocardial infarction may be influenced considerably by acute reperfusion, which is an important factor that should be incorporated in the interpretation of enzymatic estimates of infarct size in human patients.

Differential expression of three types of nitric oxide synthase in both infarcted and non-infarcted left ventricles after myocardial infarction in the rat

In the present report we investigated the differential expression of three types of nitric oxide synthase (NOS) in the left ventricle after myocardial infarction in rats. One, 3, 7, 14, 28 and 56 days (n=6-12 for each group) after ligation of a coronary artery, tissue samples were obtained from infarcted and non-infarcted tissues. The mRNA and protein levels of neuronal (n) NOS, endothelial (e) NOS and inducible (i) NOS were sequentially determined by semi-quantitative reverse transcription-polymerase chain reaction and Western blotting. Progressive left ventricular dilatation and gradual reduction in fractional shortening were confirmed by echocardiography. The expression levels of nNOS were significantly increased 1, 3 and 7 days post-infarct compared to those of sham-operated rats in both the infarcted (P<0.01) and non-infarcted regions (P<0.01). Immunohistochemical analysis showed that nNOS was localized in nerve fibers in the left ventricle and that the number of positive fibers after myocardial infarction had increased compared to that in sham-operated rats. With regard to eNOS, no significant changes in expression levels were detected between infarcted hearts and sham-operated controls. The level of iNOS expression peaked three days post-infarct and then decreased in the infarcted tissue, whereas it increased one day post-infarct, peaked at 14 and 28 days post-infarct and was still elevated in the chronic stage in the ventricular septum. iNOS immunoreactivity was detected in spared cardiomyocytes and macrophages in the infarcted region, and in cardiomyocytes in the ventricular septum. The expressions of three types of NOS were differentially regulated and iNOS produced in the non-infarcted region may contribute to the progression of heart failure after myocardial infarction in rats.

U-61, 431F, a stable prostacyclin analogue, inhibits the proliferation of bovine vascular smooth muscle cells with little antiproliferative effect on endothelial cells

The effects of U-61,431F, ciprostene, a stable prostacyclin analogue, were examined on the proliferation of cultured quiescent bovine aortic endothelial cells (EC) and smooth muscle cells (SMC). After stimulation with 5% fetal calf serum, U-61,431F suppressed both the DNA synthesis and proliferation of SMC dose-dependently at the concentration of 3-100 microM, but had no effect on either of them in EC at a concentration of up to 30 microM. The inhibitory effect on DNA synthesis was greater in SMC than in EC at 3-50 microM. When SMC were stimulated with platelet-derived growth factor (PDGF) for 2 hrs followed by a 22-hr incubation with insulin, U-61,431F (1-50 microM) administered at the time of PDGF stimulation did not inhibit DNA synthesis. SMC initiated and terminated DNA synthesis at about 15-18 h and 24 h after stimulation with serum, respectively. Inhibition of DNA synthesis in serum-stimulated SMC as a function of the addition time of U-61,431F reduced at 3-12 h after the stimulation. U-61,431F raised the cyclic AMP (cAMP) content in SMC. Moreover, a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and a more specific cAMP phosphodiesterase inhibitor, Ro 20-1724, augmented the inhibition of DNA synthesis in SMC concomitant with further elevation of cAMP level. These results suggest that U-61,431F inhibits DNA synthesis of SMC acting in the progression stage rather than in the competence stage, with little antiproliferative effect on EC. cAMP may play an important role in its antiproliferative action in SMC.

Lysophosphatidylcholine Inhibits Endothelium-Dependent Hyperpolarization and Nω-Nitro-l-Arginine/IndomethacinResistant Endothelium-Dependent Relaxation in the Porcine Coronary Artery

BACKGROUND Oxidized LDL and lysophosphatidylcholine (LPC) have been reported to inhibit the endothelium-dependent relaxation (EDR) mediated by nitric oxide. Recently, a new vasorelaxing factor, endothelium-derived hyperpolarizing factor (EDHF), which hyperpolarizes and relaxes the porcine coronary artery in the presence of N omega-nitro-L-arginine (NNA) and indomethacin (IM), has been reported. We examined whether LPC also inhibits both the EDHF-mediated relaxation and membrane hyperpolarization of the porcine coronary artery. METHODS AND RESULTS EDHF was evaluated as the bradykinin- or A23187-induced relaxation of the porcine coronary artery contracted by prostaglandin F2 alpha in the presence of NNA and IM. We also directly measured the membrane potential of the porcine coronary artery. The effects of LPC on both relaxation and membrane hyperpolarization were investigated. At concentrations of 0 to 20 mumol/L, LPC dose-dependently inhibited the NNA/IM-resistant EDR induced by bradykinin and A23187, and the relaxation was reversible after the absorption of LPC with albumin. LPC also inhibited the bradykinin- and A23187-induced hyperpolarization of the porcine coronary artery. CONCLUSIONS In the present study, LPC was found to inhibit not only nitric oxide-mediated but also EDHF-mediated relaxation of the porcine coronary artery. Our findings suggest a new regulatory mechanism in the atherosclerotic coronary artery.