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reperfusion
The 2nd Annual Scientific Meeting
009
•
JHFS
0]0
THE UNKNOWN ALTERNATIVE SPLICING GENERATES A NOVEL ISOFORM OF HUMAN ANGIOTENSIN-CONVERTING ENZYME AS A SOLUBLE FORM
EFFECTS OF ANGIOTENSIN CONVERTING ENZYME INHIBITOR ON CARDIOPULMONARY BAROREFLEX SENSITIVITY IN PATIENTS WrTH ACUTE MYOCARDIAL INFARCTION
KEIJI YONEYA, HIROSHI OKAMOTO, SATORU CHIBA, IZUMI NAKAGAWA, HIDEKI KUMAMOTO, MASASHI WATANABE, AKIRA KITABATAKE Department of Cardiovascular Medicine, Hokkaido University, Sapporo 060-8638, Japan
Makoto Hikosaka, Fumio Yuasa, Reisuke ~yama, Tetsuro Sugiura, Toshiji Iwasaka Cardiovascular Center, The Second Department of Internal Medicine, Kansal Medical University Hospital, Osaka, Japan
Human angiotensin-converting enzyme (ACE) gene exists in the chromosome 17, and two kinds of molecules are produced from the same gene locus according to the alternative splicing as the somatic and testis form. It is said theACE activity in serum derives from proteolytic release of the somatic ACE located in endothelial cell surface. V~ report here, in human somaticACE mRNA, the unrecognized alternative splicing occurs in common, and as a result, a new isoform of ACE is generated. The samples were Jurkat cell (humanT cell leukemia line) and peripheral blood leukocytes obtained healthy subjects.The mRNA was extracted from these cells, and RT-PCR was executed with the primer set in the 15th exon and the 18th exon.The electroophoresis of the PCR product visualized two bands of DNA fragment; one was the expected band derived from somaticACE mRNA, and the other was the unexpected, 150bp-longer one. This longer DNA fragment was subcloned and sequenced. As a result, the structure of this fragment was revealed to be "exon15-exon16-exon17-intronf7-exon18" of human ACE gene, and a novel alternative splicing was confirmed.This alternative splicing arose a new stop codon immediately after the 17th exon, and it was surmised that a novel isoform of ACE was £1enerated as a soluble form which deleted the transmembrane portion and C domain of two active centers of somaticACE. This isoform mRNAwas also detected in lung, kidney, aorta and pancreas, and not detected in brain, heart, liver, skeletal muscle, testis and placenta by screening the human cDNA library of each organ. In leukocytes and pancreas, the expression level was about 20 percent of somaticACE, and much lower in lung, kidney and aorta. The other research group have reported that the transgenic mouse of the equivalent artificial molecule had shown a similar phenotype of theACE-knockout mouse, therefore this spontaneously expressed molecule in human is also expected to have similar functions in vivo. Further, we have investigated the relationship between this alternative splicing and theACE Insertion/Deletion poiymorphism in the 16th intron. At this moment, any correlation have been found within both phenomena.
Cardiopulmonary baroreflex (CP) tonically inhibit sympathetic outflow and are blunted in patients with congestive heart failure. However, no data are available about the effects of angiotensin converting enzyme (ACE) inhibition on CP in patients with acute myocardial infarction (M[). The aim of the study was to evaluate the effects of ACE inhibitor (quinapril) on CP in patients with uncomplicated acute MI. Fifteen patients with uncomplicated acute M[ (Quinapdl group) underwent CP sensitivity evaluation 5 days after onset of acute MI and after 10 days of quinapril therapy. Fifteen additional acute MI patients (Control group) were evaluated at the same time interval before and after placebo administration to identify spontaneous CP variation.CP was assessed by the response of forearm vascular resistance induced by lowering central venous pressure through lower body negative pressure to -10 mmHg. Before quinapril administration, there were no significant differences in hemodynamic vadables, ejection fraction (59_+10 vs 59_+11%), CP (17_+7 vs 14_+6%), plasma noradrenaline (416_+200 vs 385_+135 pg/ml) and plasma renin activity (1.9-+ 1.6 vs 2.1 _+1.4 ng/ml/hr) between quinapril group and control group. After quinapdl administration, CP in quinapril group was significantly higher than that in control group (58_+36 vs 34+_13 %, p<0.05). Moreover, the increase in CP was larger in quinapril group than that in control group (41_+35 vs 20-+10 %, p<0.05), in quinapril group, plasma noradrenaline decreased from 416 to 331 pg/ml and plasma renin activity increased from 1.9 to 6.5 ng/ml/hr, but not in the control group. Quinapril improved CP in patients with uncomplicated acute MI. This improvement was associated with a reduction in sympathetic outflow and may contribute to the beneficial effects of ACE inhibitors in patients with uncomplicated acute M[.
011 ACE inhibitors vs Angiotensin fl receptor antagonists: Norepinephrine Release and Arrhythmias in Myocardial Ischemia/Reperfusion Eiichiro Hatta, Y o s h i r o Matsui, R o b e r t o Levi*, K e i s h u Yasuda,Department of Cardiovascular Surgery Hokkaido University School of Medicine, Sapporo 060-8648, Japan.* Department of Pharmacology, Cornell U niversity Medical College, New York, NY 10021, USA Excessive NE release causes ischemic cardiac dysfunction and arrhythmias. By acting atAT1 and B2-receptors(R), exogenous angiotensin II (All) and bradykinin(BK) enhances NE release and associated arrhythmias (VF) in ischemia/reperfusion (I/R). SinceAII and BK production increases in the ischemic heart andACE inhibitors alter both of them, we have assessed how endogenousAII and BK contribute to NE release and VE After 10-rain global ischemia in isolated guinea-pig hearts, exocytotic NE release (ER) (10 pmol/g) occurred in the absence of VE In contrast, 20-min global ischemia caused carrier-mediated NE release (CMR) (800pmol/g) and 2-min VF. To clarify the role of endogenousAII and BK in NE release and VF, we inhibited IocalAII and BK production. The serine protease inhibitors aprotinin (500 KIU/ml) and SBTI (100 #g/roll decreased NE release (50%, both ER and CMR) and abolished VE Selective AT1-R antagonist EXP3174 (100nM) attenuated ER, CMR and VF by 30, 35 and 50%, respectively. Although selective B2-R antagonist HOE140 (30nM) altered neither NE release nor VF, EXP3174 and HOE140 in combination decreased ER, CMR and VF by 50, 60 and 60%, respectively However, kininase II/ACE inhibitor enalaprilat in combination with the kininase I inhibitor MERGETPA(1 #M of each), increased ER 10-fold and CMR by 50%. VF lasted 5 times as long. These effects were blocked by HOE 140. Thus, in myocardial ischemia All is locally produced in amounts sufficient to enhance ER, CMR and reperfusion arrhythmias via AT1-R. Although local BK production increases in myocardial ischemia, the elfects of BK on adrenergic nerve terminals are uncovered only when BK half-life is prolonged and/or whenAII effects are suppressed.
012 COMPARATIVE EFFECTS OF CANDESARTAN CILEXITIL AND CILAZAPRIL ON CARDIAC FUNCTION AND GENE EXPRESSION IN MYOCARDIAL INFARCTED RATS Minoru Yoshiyama, Kazuhide Takeuchi, Takashi Omura, Itiroyuld Yamagishi, Iku Toda, Masakazu Teragald, Kaname Akioka, Junichi Yoshikawa First Department of Internal Medicine, Osaka City University Medical School, Osaka 545-0051, Japan
The purpose of this study was to assess the comparative effect of angiotensin II receptor antagonist, candesartan cilexitil (1,10 mg/kg/day), and angiotensin converting enzyme inhibitor (ACEI), cilazapril (1, 10 mg/kg/day), on left ventricular systalic and diastolic function in myocardial infarcted rats by using Dopplerechocardiography. On 1 and 4 weeks after myocardial infarction, cardiac function was measured and mRNAs in nonischemic myocardium were analyzed. Left ventricular end-diastolic dimension (LVDd) and fractional shortening on 1 week were not different from treated groups, candesartan at 1 mg/kg/day and cilazapril at 1 mg/kg/day prevented an increase of LVDd and fractional shortening on 4 weeks at same extent. E/A wave velocity ratio (E/A) and the rate of E wave deceleration (E dec.) increased to 9.5+2.2 and 26.3_+2.6 m/s 2 on 1 week after MI. Candesartan cilexitil and cilazapril prevented the increase of E/A (4.2-+0.5;P<0.01, 4.9_+0.4;P<0.01) and E dec. (15.3_+2.2 m/s2;p<0.01, 16.3_+3.2 m/s2;p<0.01) on 1 week after MI, and those on 4 weeks after MI(P<0.01). The gene expressions of 6myosin heavy chain (MHC), o~-skeletal actin, atrial natduretic peptide (ANP), collagen I and III in the non-ischemic left ventdcular myocardium increased by 1.8-, 2.9-, 4.2-, 3.0-, and 2.6-fold on 1 week, respectively (P<0.01) and RV also increased. Candesartan and cilazapdl significantly suppressed fetal gene expressions of nonischemic left and right ventricles on 1 week. Both drugs at 10 mg/kg/day has same effect on cardiac function and gene expression. In conclusion, candesartan and cilazapril prevented diastolic dysfunction after myocardial infarction prior to improve systolic function and both drugs showed same effect on cardiac function and cardiac gene expression.
67
009
•
JHFS
0]0
THE UNKNOWN ALTERNATIVE SPLICING GENERATES A NOVEL ISOFORM OF HUMAN ANGIOTENSIN-CONVERTING ENZYME AS A SOLUBLE FORM
EFFECTS OF ANGIOTENSIN CONVERTING ENZYME INHIBITOR ON CARDIOPULMONARY BAROREFLEX SENSITIVITY IN PATIENTS WrTH ACUTE MYOCARDIAL INFARCTION
KEIJI YONEYA, HIROSHI OKAMOTO, SATORU CHIBA, IZUMI NAKAGAWA, HIDEKI KUMAMOTO, MASASHI WATANABE, AKIRA KITABATAKE Department of Cardiovascular Medicine, Hokkaido University, Sapporo 060-8638, Japan
Makoto Hikosaka, Fumio Yuasa, Reisuke ~yama, Tetsuro Sugiura, Toshiji Iwasaka Cardiovascular Center, The Second Department of Internal Medicine, Kansal Medical University Hospital, Osaka, Japan
Human angiotensin-converting enzyme (ACE) gene exists in the chromosome 17, and two kinds of molecules are produced from the same gene locus according to the alternative splicing as the somatic and testis form. It is said theACE activity in serum derives from proteolytic release of the somatic ACE located in endothelial cell surface. V~ report here, in human somaticACE mRNA, the unrecognized alternative splicing occurs in common, and as a result, a new isoform of ACE is generated. The samples were Jurkat cell (humanT cell leukemia line) and peripheral blood leukocytes obtained healthy subjects.The mRNA was extracted from these cells, and RT-PCR was executed with the primer set in the 15th exon and the 18th exon.The electroophoresis of the PCR product visualized two bands of DNA fragment; one was the expected band derived from somaticACE mRNA, and the other was the unexpected, 150bp-longer one. This longer DNA fragment was subcloned and sequenced. As a result, the structure of this fragment was revealed to be "exon15-exon16-exon17-intronf7-exon18" of human ACE gene, and a novel alternative splicing was confirmed.This alternative splicing arose a new stop codon immediately after the 17th exon, and it was surmised that a novel isoform of ACE was £1enerated as a soluble form which deleted the transmembrane portion and C domain of two active centers of somaticACE. This isoform mRNAwas also detected in lung, kidney, aorta and pancreas, and not detected in brain, heart, liver, skeletal muscle, testis and placenta by screening the human cDNA library of each organ. In leukocytes and pancreas, the expression level was about 20 percent of somaticACE, and much lower in lung, kidney and aorta. The other research group have reported that the transgenic mouse of the equivalent artificial molecule had shown a similar phenotype of theACE-knockout mouse, therefore this spontaneously expressed molecule in human is also expected to have similar functions in vivo. Further, we have investigated the relationship between this alternative splicing and theACE Insertion/Deletion poiymorphism in the 16th intron. At this moment, any correlation have been found within both phenomena.
Cardiopulmonary baroreflex (CP) tonically inhibit sympathetic outflow and are blunted in patients with congestive heart failure. However, no data are available about the effects of angiotensin converting enzyme (ACE) inhibition on CP in patients with acute myocardial infarction (M[). The aim of the study was to evaluate the effects of ACE inhibitor (quinapril) on CP in patients with uncomplicated acute MI. Fifteen patients with uncomplicated acute M[ (Quinapdl group) underwent CP sensitivity evaluation 5 days after onset of acute MI and after 10 days of quinapril therapy. Fifteen additional acute MI patients (Control group) were evaluated at the same time interval before and after placebo administration to identify spontaneous CP variation.CP was assessed by the response of forearm vascular resistance induced by lowering central venous pressure through lower body negative pressure to -10 mmHg. Before quinapril administration, there were no significant differences in hemodynamic vadables, ejection fraction (59_+10 vs 59_+11%), CP (17_+7 vs 14_+6%), plasma noradrenaline (416_+200 vs 385_+135 pg/ml) and plasma renin activity (1.9-+ 1.6 vs 2.1 _+1.4 ng/ml/hr) between quinapril group and control group. After quinapdl administration, CP in quinapril group was significantly higher than that in control group (58_+36 vs 34+_13 %, p<0.05). Moreover, the increase in CP was larger in quinapril group than that in control group (41_+35 vs 20-+10 %, p<0.05), in quinapril group, plasma noradrenaline decreased from 416 to 331 pg/ml and plasma renin activity increased from 1.9 to 6.5 ng/ml/hr, but not in the control group.
011 ACE inhibitors vs Angiotensin fl receptor antagonists: Norepinephrine Release and Arrhythmias in Myocardial Ischemia/Reperfusion Eiichiro Hatta, Y o s h i r o Matsui, R o b e r t o Levi*, K e i s h u Yasuda,Department of Cardiovascular Surgery Hokkaido University School of Medicine, Sapporo 060-8648, Japan.* Department of Pharmacology, Cornell U niversity Medical College, New York, NY 10021, USA Excessive NE release causes ischemic cardiac dysfunction and arrhythmias. By acting atAT1 and B2-receptors(R), exogenous angiotensin II (All) and bradykinin(BK) enhances NE release and associated arrhythmias (VF) in ischemia/reperfusion (I/R). SinceAII and BK production increases in the ischemic heart andACE inhibitors alter both of them, we have assessed how endogenousAII and BK contribute to NE release and VE After 10-rain global ischemia in isolated guinea-pig hearts, exocytotic NE release (ER) (10 pmol/g) occurred in the absence of VE In contrast, 20-min global ischemia caused carrier-mediated NE release (CMR) (800pmol/g) and 2-min VF. To clarify the role of endogenousAII and BK in NE release and VF, we inhibited IocalAII and BK production. The serine protease inhibitors aprotinin (500 KIU/ml) and SBTI (100 #g/roll decreased NE release (50%, both ER and CMR) and abolished VE Selective AT1-R antagonist EXP3174 (100nM) attenuated ER, CMR and VF by 30, 35 and 50%, respectively. Although selective B2-R antagonist HOE140 (30nM) altered neither NE release nor VF, EXP3174 and HOE140 in combination decreased ER, CMR and VF by 50, 60 and 60%, respectively However, kininase II/ACE inhibitor enalaprilat in combination with the kininase I inhibitor MERGETPA(1 #M of each), increased ER 10-fold and CMR by 50%. VF lasted 5 times as long. These effects were blocked by HOE 140. Thus, in myocardial ischemia All is locally produced in amounts sufficient to enhance ER, CMR and reperfusion arrhythmias via AT1-R. Although local BK production increases in myocardial ischemia, the elfects of BK on adrenergic nerve terminals are uncovered only when BK half-life is prolonged and/or whenAII effects are suppressed.
012 COMPARATIVE EFFECTS OF CANDESARTAN CILEXITIL AND CILAZAPRIL ON CARDIAC FUNCTION AND GENE EXPRESSION IN MYOCARDIAL INFARCTED RATS Minoru Yoshiyama, Kazuhide Takeuchi, Takashi Omura, Itiroyuld Yamagishi, Iku Toda, Masakazu Teragald, Kaname Akioka, Junichi Yoshikawa First Department of Internal Medicine, Osaka City University Medical School, Osaka 545-0051, Japan
The purpose of this study was to assess the comparative effect of angiotensin II receptor antagonist, candesartan cilexitil (1,10 mg/kg/day), and angiotensin converting enzyme inhibitor (ACEI), cilazapril (1, 10 mg/kg/day), on left ventricular systalic and diastolic function in myocardial infarcted rats by using Dopplerechocardiography. On 1 and 4 weeks after myocardial infarction, cardiac function was measured and mRNAs in nonischemic myocardium were analyzed. Left ventricular end-diastolic dimension (LVDd) and fractional shortening on 1 week were not different from treated groups, candesartan at 1 mg/kg/day and cilazapril at 1 mg/kg/day prevented an increase of LVDd and fractional shortening on 4 weeks at same extent. E/A wave velocity ratio (E/A) and the rate of E wave deceleration (E dec.) increased to 9.5+2.2 and 26.3_+2.6 m/s 2 on 1 week after MI. Candesartan cilexitil and cilazapril prevented the increase of E/A (4.2-+0.5;P<0.01, 4.9_+0.4;P<0.01) and E dec. (15.3_+2.2 m/s2;p<0.01, 16.3_+3.2 m/s2;p<0.01) on 1 week after MI, and those on 4 weeks after MI(P<0.01). The gene expressions of 6myosin heavy chain (MHC), o~-skeletal actin, atrial natduretic peptide (ANP), collagen I and III in the non-ischemic left ventdcular myocardium increased by 1.8-, 2.9-, 4.2-, 3.0-, and 2.6-fold on 1 week, respectively (P<0.01) and RV also increased. Candesartan and cilazapdl significantly suppressed fetal gene expressions of nonischemic left and right ventricles on 1 week. Both drugs at 10 mg/kg/day has same effect on cardiac function and gene expression. In conclusion, candesartan and cilazapril prevented diastolic dysfunction after myocardial infarction prior to improve systolic function and both drugs showed same effect on cardiac function and cardiac gene expression.
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