심방이뇨호르몬 분비와 지방대사에 있어서 안지오텐신의 병태생리학적 역할

다국어 초록 (Multilingual Abstract)

Angiotensin II (Ang II) and angiotensin-(1-7) [Ang-(1-7)] are primary effector molecules in renin-angiotensin system and cause a variety of functions in body fluid metabolism. Atrial natriuretic peptide (ANP) is related with both two peptides. However, the roles of angiotensin peptides on ANP secretion are still controversial. The aim of the present study is to investigate the roles of Ang II and Ang-(1-7) on ANP secretion and metabolism in physiological and pathological conditions.
An isolated perfused beating atria model and radioimmunoassay were used. In vivo experiments were also performed in Sprague-Dawley rats and mice. Radioimmunoassay, real-time polymerase chain reaction, western blot analysis, and histological analysis were used. Ang II decreased high stretch-induced ANP secretion and concentration in isolated perfused beating atria. Antagonist of Ang II receptor type 1 (AT1R) not only suppressed the effect of Ang II, but also had a self-stimulatory effect on ANP secretion. In two-kidney, one-clipped (2K1C) hypertensive rats, the effects of Ang II and AT1R antagonist on ANP secretion were more accentuated.
On the other hand, a disruption of caveolae with methyl-？-cyclodextrin (MbCD) caused abolishment of the effects of Ang II and AT1R antagonist in high-stretched condition. In the presence of MbCD, the proportion of proANP to ANP in secreted ANP was increased. These results indicated : i) Ang II generated endogenously by atrial stretch suppressed stretch-induced ANP secretion via AT1R, and ii) disruption of caveolae altered AT1R-mediated Ang II effect on ANP secretion and its effect might be mediated by processing of proANP.
Next, the role of Ang-(1-7) on lipolysis was investigated. Ang-(1-7) increased glycerol release and pretreatment with antagonist of Mas R or inhibitors of phosphatidyl inositol 3-kinase (PI3K) and nitric oxide synthase (NOS) blocked lipolytic effect of Ang-(1-7). Elevated plasma Ang-(1-7) level induced by chronic administration of angiotensin converting enzyme (ACE) inhibitor caused decrease of weight gain, but did not cause adipocyte hypotrophy. These results indicated that Ang-(1-7) stimulated lipolysis through activation of Mas/PI3K/eNOS signaling, and its effect might not relate to adipocyte hypertrophy.
It has been reported that Ang II and AT1R related with Januse kinase (JAK)/signal transducers and activators of transcription protein (STAT) signaling are involved in the pathogenesis of I/R injury. In the present study, the role of JAK3 on I/R injury was investigated. Increases of I/R-induced plasma creatinine phosphokinse (CPK) and lactate dehydrogenase (LDH) levels, infarct area, and apoptosis were decreased in JANEX-1 (a selective JAK3 inhibitor) treated-mice and JAK3 -/- mice. I/R-induced reduction of left ventricular function was improved in both groups. The expression of JAK2/STAT3 signaling was increased by inhibition of JAK3. These data indicated that inhibition of JAK3 improves cardiac functions and injury in I/R and its effect may be due to activation of cardioprotective JAK2/STAT3 pathway.
In conclusion, these results suggest that endogenous Ang II suppresses stretch-induced ANP secretion via AT1R and disruption of caveolae alters an effect of Ang II. Ang-(1-7) stimulates lipolysis through Mas R/PI3K/eNOS signaling and inhibition of JAK3 improves cardiac function and injury against I/R. So, Ang II and Ang-(1-7) have critical antagonistic roles in cardiovascular physiology and lipid metabolism.

목차 (Table of Contents)

• Chapter I. Introduction 1
• 1. Atrial natriuretic peptide 1
• 2. Renin-angiotensin system 2
• 3. The caveolae 4
• 4. Role of RAS in lipolysis 5
• 5. Mechanism of myocardial ischemia/reperfusion injury 6
• 6. Objective 8
• Chapter II. Materials and Methods 9
• 1. Chemicals 9
• 2. Animals 9
• 3. Animal models 10
• 3.1 Two-kidney, one-clipped hypertensive rat model 10
• 3.2 Myocardial I/R mouse model 10
• 3.3 Isolated perfused beating atrial model 11
• 3.4 Isolated primary adipocytes 13
• 4. Experimental protocols 13
• 4.1 Effect of Ang II on stretch-induced ANP secretion 13
• 4.2 The involvement of caveolae in regulation of stretchinduced and Ang IIAng II type 1 receptormediated ANP secretion 15
• 4.3 Effect of Angiotensin-(1-7) on lipolysis 16
• 5. Chronic infusion of chemicals using mini-osmotic pump 17
• 6. Measurement of ANP concentration 17
• 7. Measurement of ECF translocation 19
• 8. Measurement of plasma Ang-(1-7) concentration 20
• 9. Real-time polymerase chain reaction 21
• 10. Western blot analysis 22
• 11. High performance liquid chromatography 23
• 12. Electron microscopy 24
• 13. Measurement of plasma LDH and CPK 24
• 14. Infarct size 24
• 15. Histological analysis 25
• 15.1 Hematoxylin and eosin staining 25
• 15.2 Immunohistochemistry 25
• 15.3 TdT mediated dUTP nick end labeling statining 26
• 16. Echocardiography 26
• 17. Statistical analysis 27
• Chapter III. Results 28
• 1. Effect of Ang II on atrial hemodynamics and stretch-induced ANP secretion 28
• 1.1 Effects of Ang II on strectch-induced atrial contractility and ANP secretion 28
• 1.2 Modulatory effects of Ang II in high strectch-induced ANP secretion with Ang II receptor antagonists 29
• 1.3 Effects of Ang II and losartan on high strectch-induced atrial parameters in hypertrophied atria from 2K1C rats 30
• 2. The involvement of caveolae in regulation of stretch- and Ang II-mediated ANP secretion 31
• 2.1 Effects of methyl--cyclodextrin pretreatment on basal and high stretch-induced atrial contractility and ANP secretion 31
• 2.2 Modulation of Ang II-induced suppression of high stretch-induced ANP secretion by MbCD pretreatment 32
• 2.3 Modulation of losartan-induced augmentation of stretch-induced ANP secretion by MbCD pretreatment 33
• 2.4 Modulation of Ang-(1-7)-induced augmentation of stretch-induced ANP secretion by MbCD pretreatment 33
• 3. Effect of Ang-(1-7) on lipolysis 34
• 3.1 Basal effect of Ang-(1-7) on lipolysis 34
• 3.2 Modulation of effects of Ang-(1-7) in lipolysis with Ang II and Ang-(1-7) receptor antagonists or inhibitors of PI3K/Akt/eNOS signaling pathway 34
• 3.3 Effect of captopril on plasma Ang-(1-7) concentration 35
• 3.4 Effect of captopril on change of body weight gain and adipose tissue weight in vivo 35
• 3.5 Protein expression on lipolytic signaling pathway 36
• 4. Effect of JAK3 on myocardial I/R injury 36
• 4.1 Effects of JAK3 in plasma LDH and CPK activity on myocardial I/R injury 36
• 4.2 Infarct size 36
• 4.3 Modulatory effects of JAK3 on cardiac function in sham-operated and I/R-operated hearts 37
• 4.4 Modulation of induction of intracellular signaling pathways by JAK 3 37
• 4.5 Effect of inhibition of JAK3 on apoptosis and cardiac injury at cellular level 38
• Chapter IV. Discussion 39
• Chapter V. References 80