nNOS activation  is involved in  insulin-mediated cardiovascular effects  in  the nucleus tractus solitarii  of rats Chian...
Introduction   › › ›  Generalization -  Receptor tyrosine kinase (RTK) -  Activation : insulin binding Location in CNS -  ...
Introduction   › › ›  1  Huang H. N. et al. 2004.  Circulation.  110: 2476-2483. Insulin receptor (IR) Insulin <ul><li>Ins...
Introduction   › › ›  The role of nucleus tractus solitarius (NTS) on cardiovascular function  1   Autonomic control of th...
Introduction   › › ›  <ul><li>The insulin-mediated signaling  in  the NTS  may   play a significant role in the regulation...
Introduction   › › ›  High expression in NTS Neuronal nitric oxide synthase (nNOS) <ul><li>The NO derived from nNOS  in  t...
Introduction   › › ›  The relationship between insulin-PI3K-Akt pathway and nNOS-derived NO in NTS  1,2   1  Huang H. N. e...
Hypothesis   › › ›  nNOS phosphorylation  is induced by  insulin injection  in  the NTS   via  PI3K-Akt signaling pathway ...
Experimental procedures  › › ›  Experimental animals Wistar-Kyoto (WKY) rats Gender Weight Male 250-300 g Hemodynamic meas...
Experimental procedures  › › ›  Western blot analysis <ul><li>Western blot analysis  is used to determine  nNOS and p-nNOS...
Experimental procedures  › › ›  Co-immunoprecipitation (Co-IP) assay <ul><li>Co-IP assay  is used to determine interaction...
Statistical analysis Experimental procedures  › › ›  Experimental data <ul><li>Mean ± SEM. </li></ul>Statistical differenc...
Results and discussion  › › ›  <ul><li>The attenuation effect of  7-NI pretreatment  lasted for  more than 90 min. </li></...
Results and discussion  › › ›  <ul><li>The attenuation effect of  vinyl-L-NIO pretreatment  reached  plateau status at 10 ...
Inhibitory effect of the nNOS inhibitor on insulin-mediated cardiovascular effect  1-5 1  Huang H. N. et al. 2004.  Circul...
Results and discussion  › › ›  A B C <ul><li>Insulin injection  significantly increased  nNOS phosphorylation at Ser 1416 ...
nNOS phosphorylation at ser 1416 induced by insulin injection in the NTS  1-3 1  Huang H. N. et al. 2004.  Circulation.  1...
<ul><li>nNOS  can be phosphorylated by  many kinases  including  PKA, PKC and CaMK II. </li></ul><ul><li>The phosphorylati...
Results and discussion  › › ›  <ul><li>The results  suggested that  P-Akt 473  interact with  P-nNOS 1416. </li></ul><ul><...
Results and discussion  › › ›  <ul><li>The results  suggested that  P-Akt 473  interact with  P-nNOS 1416. </li></ul><ul><...
Results and discussion  › › ›  <ul><li>The results  suggested that  P-Akt 473  interact with  P-nNOS 1416. </li></ul><ul><...
Summary   › › ›  Insulin receptor (IR) Insulin nNOS activation is involved in insulin-mediated cardiovascular effect in th...
Summary   › › ›  nNOS activation is involved in insulin-mediated cardiovascular effect in the NTS nNOS   is  downstream fa...
Summary   › › ›  nNOS activation is involved in insulin-mediated cardiovascular effect in the NTS Hypotension Bradycardia ...
Summary   › › ›  nNOS activation is involved in insulin-mediated cardiovascular effect in the NTS Hypertension Tachycardia...
Thank You for Your Attention !!
Wistar-Kyoto (WKY) rats <ul><li>Coat color:  white (albino) </li></ul><ul><li>Behavior:  </li></ul><ul><li>Sensitive to th...
1  Julian F.R.P. 1999.  Exp physiol.  84: 815-833;  2  Andresen M. C. and Kunze D. L. 1994.  Annu. Rev. Physiol . 56: 93-1...
Backup  › › ›  Integrated neural pathway of NTS  1 1   Libby. Braunwald's Heart Disease, 8 th  ed: 2007
1  Klabunde R. E.  Cardiovascular Physiology Concepts . Philadelphia: Lippincott Williams & Wilkins, 2005 Backup  › › ›  T...
1  subsites.egaa-gym.dk/nr/biologi/roholt/Noter/note21a.html;  2  w ww.med.cmu.ac.th/dept/pharmaco/pharmacology/lesson01/0...
1  Kevin J. T. 2009.  Nature Reviews Immunology  9: 418-428. Backup  › › ›  The role of NTS on inflammatory reflex  1 Abbr...
1  Ravinder K. et al. 2006.  GI Motility online.  doi:10.1038/gimo14   Backup  › › ›  The role of NTS on swallowing  1 Mec...
Backup  › › ›  Nitric oxide (NO) and NO synthase (NOS)  1,   2   Precursor Biosynthesis  Distribution Chemical compound <u...
Backup  › › ›  1 Richard Bruckdorfer. 2005. Molecular Aspects of Medicine 26:3-31 Structure and function of NOS isoforms  ...
Backup  › › ›  nNOS phosphorylation  1-4   1  Song T. et al. 2004.  FEBS Letters . 570 :133–137  2  Rameau G. A. et al. 20...
Backup  › › ›  Comparison of 7-NI and vinyl-L-NIO NADPH  FAD FMN Zn 2+ /BH 4 /Heme CaM COOH NH 2 L-arginine L-citrulline +...
1  Herring N. and Paterson D. J. 2001.  J Physiol . 535: 507-18 2  Paton J. F. R. et al. 2002.  TRENDS in Neuroscience . 2...
1  Toda N. and Ayajiki K. 2010.  Alcohol and Alcoholism . 45 (4): 347-355 Backup  › › ›  The role of nNOS-derived NO on bl...
Abbreviation:  PIP 2  = Phosphatidylinositol (PI) 4,5-bisphosphate, PIP 3  = PI 3,4,5-triphosphate  Structure and classifi...
Morpholine derivetive Reversible Competitive Cell-permeable <ul><li>LY294002  inhibits  ATP binding site  on  the catalyti...
Backup  › › ›  3-phosphoinositide dependent protein kinase (PDK)  1 1  Reuben J. et al. 2006.  Nature.  441: 424-430  PDK1...
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nNOS activation is involved in insulin-mediated CV effects in the NTS

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  • Insulin เป็น peptide hormone มี M.W. = 5805 Da และประกอบไปด้วย 51 amino acid
  • Insulin เป็น peptide hormone มี M.W. = 5805 Da และประกอบไปด้วย 51 amino acid
  • Chemoreceptor มี 2 ประเภท คือ peripheral chemoreceptor (carotic กับ aortic body) และ central chemoreceptor (medulla) Baroreceptor อยู่ที่ carotic sinus และ aortic arch
  • NTS is located along the length of the medulla (with a small portion in the lower pons ) Chemoreceptor มี 2 ประเภท คือ peripheral chemoreceptor (carotic กับ aortic body) และ central chemoreceptor (medulla) Baroreceptor อยู่ที่ carotic sinus และ aortic arch Inotrophy : contractility; chronotrophy : heart rate and Inotrophy : conduction velocity
  •   Baroreceptors อยู่ที่ aortic arch และ carotid sinus เป็น receptor ที่ถูกกระตุ้นด้วยความดัน ( pressure) ดังนั้นกรณีที่หลอดเลือดหดตัว ( vasoconstriction) จะทำให้ baroreceptor ถูกกระตุ้นได้มาก ซึ่งจะยังผลให้ vagal centers และ inhibitory interneurons ในสมองส่วน medulla ถูกกระตุ้นได้มากขึ้น ทำให้  - Vagus nerve ถูกกระตุ้น ทำให้ระบบประสาท parasympathetic ที่หัวใจทำงานมาก หัวใจเต้นช้าลง - Vasomotor center (VMC) ทำงานได้น้อยลง เนื่องจาก inhibitory interneurones ถูกกระตุ้นมาก ส่งผลให้ลดกระแสประสาทจาก sympathetic ไปยังหลอดเลือดและหัวใจ
  • RVLM เป็นที่อยู่ของ sympathoexcitatory neurons ส่วน NA เป็นที่อยู่ของ vagal motor neuron
  • การกลืนสามารถแบ่งออกได้เป็น 3 ระยะคือ ระยะช่องปาก (oral phase) ระยะคอหอย ( pharyngeal phase) และระยะหลอดอาหาร ( esophageal phase) ระยะช่องปาก ( oral phase) และคอหอย ( pharyngeal phase) จะต่อเนื่องกันและอาหารที่ผ่าน 2 ระยะนี้จะใช้เวลาสั้นมาก Crural diaphragm เป็น tendinous structures ของ diaphragm
  • Ca 2+ /calmodulin (CaM)-dependent kinase มีชื่อย่อว่า CaM-K nNOS ถูก dephosphorylate ด้วย phosphatase calcineurin ที่ serine 847
  • 7-NI interacts competitively at the substrate binding site and the co-factor tetrahydrobiopterin (BH4) in the NOS enzyme
  • Pacemaker cell ส่วนใหญเป็น sinoatrial (SA) node Muscarinic type 2 acetylcholine receptor ที่กระตุ้นการทำงานของ G i protein
  • Pacemaker cell ส่วนใหญเป็น sinoatrial (SA) node Muscarinic type 2 acetylcholine receptor ที่กระตุ้นการทำงานของ G i protein
  • The p85 subunit of PI3K contains SH2 and SH3 domain The SH2 domain binds phosphotyrosine residues
  • LY294002 is a morpholine derivative of quercetin (flavonoid) LY294002 is a cell-permeable compound LY294002 acts as a potent and selective inhibitor of PI3K
  • 7-NI interacts competitively at the substrate binding site and the co-factor tetrahydrobiopterin (BH4) in the NOS enzyme
  • nNOS activation is involved in insulin-mediated CV effects in the NTS

    1. 1. nNOS activation is involved in insulin-mediated cardiovascular effects in the nucleus tractus solitarii of rats Chiang H.T. et al. 2009 Neuroscience I.F. = 3.556
    2. 2. Introduction › › › Generalization - Receptor tyrosine kinase (RTK) - Activation : insulin binding Location in CNS - Cerebellum - Olfactory bulb - Hippocampus - Hypothalamus - Cerebral cortex - Nucleus tractus solitarius CVS function - Heart rate - Arterial blood pressure 1 Unger J. W. et al. 1991. Prog Neurobiol. 36: 343-362; 2 Adamo M. et al. 1989. Mol Neurobiol . 3:71-100. The insulin receptor in the central nervous system (CNS) 1,2 Insulin receptor Generalization CVS function Location in CNS
    3. 3. Introduction › › › 1 Huang H. N. et al. 2004. Circulation. 110: 2476-2483. Insulin receptor (IR) Insulin <ul><li>Insulin-PI3K-Akt-NOS signaling pathway plays an important role in central cardiovascular regulation </li></ul><ul><li>Bradycardia is the resting heart rate of under 60 beats per minute </li></ul>The role of insulin receptor in the CNS on cardiovascular function 1 P P P P IR substrate IR substrate PI3K P PI3K P PDK P PDK P Akt P P Akt P P NOS-derived NO ↓ Heart rate ↓ Arterial pressure Hypotension Bradycardia Insulin-Pi3K-Akt pathway PIP 2 P P PIP 2 P P PIP 3 P P P PIP 3 P P P
    4. 4. Introduction › › › The role of nucleus tractus solitarius (NTS) on cardiovascular function 1 Autonomic control of the cardiovascular system Arterial baroreceptor Chemoreceptor Cardiopulmonary receptor Visceral receptor Nucleus tractus solitarius Primary integrative center 1 Dampney R. A. 1994. Physiol Rev. 74: 323-363 Sympathetic nerve activity Heart rate (HR) Blood pressure (BP)
    5. 5. Introduction › › › <ul><li>The insulin-mediated signaling in the NTS may play a significant role in the regulation of cardiovascular activity </li></ul>The role of insulin receptor in NTS on cardiovascular function 1 Akt phosphorylation Insulin receptor activation Insulin microinjection into NTS Predominant depressor Bradycardic effect 1 Huang H. N. et al. 2004. Circulation. 110: 2476-2483.
    6. 6. Introduction › › › High expression in NTS Neuronal nitric oxide synthase (nNOS) <ul><li>The NO derived from nNOS in the NTS contributes to control cardiovascular effect . </li></ul>The role of nNOS in NTS on cardiovascular function 1-6 1 Tseng C. J. et al. 1996. Hypertension. 2 7: 36-42; 2 Lo W. C. et al. 2004. Hypertension. 2 2: 36-42 3 Zhang K. et al. 1998. Brain Res. 786 : 219-225; 4 Bredt D. S. et al. 1990. Nature. 347: 768-770 5 Maeda M. et al. 1999. Neuroreport. 10: 1957-1960; 6 Ollerstam A. et al. 1997. J Clin Invest. 99 : 2212-2218 Nitric oxide (NO) production L-arginine L-arginine microinjection <ul><li>nNOS inhibitor: </li></ul><ul><li>7-nitroindazole (7-NI) </li></ul><ul><li>nNOS antisense oligonucleotide: </li></ul>Inhibition Blood pressure Prominent depressor Bradycardic effect <ul><li>Sympathetic outflow from brainstem </li></ul><ul><li>Vasomotor tone </li></ul><ul><li>Blood pressure </li></ul>Regulate Cardiovascular function
    7. 7. Introduction › › › The relationship between insulin-PI3K-Akt pathway and nNOS-derived NO in NTS 1,2 1 Huang H. N. et al. 2004. Circulation. 110: 2476-2483.; 2 Bredt D. S. et al. 1990. Nature. 347: 768-770 Control Control The relationship between insulin-PI3K-Akt signaling pathway and nNOS-derived NO in NTS is unclear
    8. 8. Hypothesis › › › nNOS phosphorylation is induced by insulin injection in the NTS via PI3K-Akt signaling pathway 1 nNOS is downstream factor in insulin-PI3K-Akt signaling pathway 2 The activation of nNOS-derived NO by insulin-PI3K-Akt signaling pathway participates in central cardiovascular regulation 3
    9. 9. Experimental procedures › › › Experimental animals Wistar-Kyoto (WKY) rats Gender Weight Male 250-300 g Hemodynamic measurement 300 mg/kg i.v. If necessary 1.0 g/kg i.p. Anesthesia - Urethane NTS microinjection Drug administration <ul><li>Insulin </li></ul><ul><li>PI3K inhibitor : </li></ul><ul><li>- LY294002 </li></ul><ul><li>nNOS inhibitor : </li></ul><ul><li>- 7-nitroindrazole (7-NI) </li></ul><ul><li>- Vinyl-L-NIO </li></ul>Measurement <ul><li>Blood pressure : </li></ul><ul><li>- Pressure transducer </li></ul><ul><li>- Polygraph </li></ul><ul><li>Heart rate : </li></ul><ul><li>- Tachograph amplifier </li></ul>
    10. 10. Experimental procedures › › › Western blot analysis <ul><li>Western blot analysis is used to determine nNOS and p-nNOS protein level before and after insulin and LY294002 (PI3K inhibitor) injection in the NTS of WKY rats. </li></ul>Immunohistochemistry analysis <ul><li>Immunohistochemical staining is used to determine nNOS phosphorylation after insulin injection in the NTS of WKY rats. </li></ul>
    11. 11. Experimental procedures › › › Co-immunoprecipitation (Co-IP) assay <ul><li>Co-IP assay is used to determine interaction between Akt and nNOS </li></ul>In vitro kinase assay <ul><li>In vitro kinase assay is used to determine Akt activity on nNOS phosphorylation </li></ul>
    12. 12. Statistical analysis Experimental procedures › › › Experimental data <ul><li>Mean ± SEM. </li></ul>Statistical difference <ul><li>P value < 0.05 </li></ul>Statistical significant <ul><li>Paired t -test </li></ul><ul><li>One-way ANOVA </li></ul><ul><li>with Dunnett ‘s test </li></ul>
    13. 13. Results and discussion › › › <ul><li>The attenuation effect of 7-NI pretreatment lasted for more than 90 min. </li></ul><ul><li>Pretreatment with 7-NI significantly increased MBP and HR to compare the control. </li></ul>Inhibitory effect of the nNOS inhibitor 7-NI on cardiovascular regulation Fig. 1 Cardiovascular effect of unilateral injection of insulin into the NTS before and after 7-NI injection in WKY rats. A B Unilateral microinjection of insulin The NTS of 8-week-old- WKY rats Into Prominent depressor Produce Bradycardic effect 7-NI pretreatment lasted for more than 90 min. lasted for more than 90 min.
    14. 14. Results and discussion › › › <ul><li>The attenuation effect of vinyl-L-NIO pretreatment reached plateau status at 10 min after insulin injection and returned to depressor and bradycardic effect at 30 or 60 min. </li></ul>A B <ul><li>Pretreatment with vinyl-L-NIO significantly increased MBP and HR to compare the control. </li></ul>Unilateral microinjection of insulin The NTS of 8-week-old- WKY rats Into Prominent depressor Produce Bradycardic effect Vinyl-L-NIO pretreatment Inhibitory effect of the nNOS inhibitor vinyl-L-NIO on cardiovascular regulation Fig. 2 Cardiovascular effect of unilateral injection of insulin into the NTS before and after vinyl-L-NIO injection in WKY rats.
    15. 15. Inhibitory effect of the nNOS inhibitor on insulin-mediated cardiovascular effect 1-5 1 Huang H. N. et al. 2004. Circulation. 110: 2476-2483; 2 Tseng C. J. et al. 1996. Hypertension. 2 7: 36-42; 3 Lo W. C. et al. 2004. Hypertension. 2 2: 36-42; 4 Ollerstam A. et al. 1997. J Clin Invest. 99 : 2212-2218; 5 Babu B. R. and Griffith O. W. 1998. J Biol Chem. 273: 8882-8889. Results and discussion › › › Insulin-induced insulin receptor activity 7-NI Vinyl-L-NIO nNOS-dependent nitric oxide Nucleus tractus solitarius function regulate Sympathetic nervous activity Baroreceptor reflex modulate Cardiovascular function
    16. 16. Results and discussion › › › A B C <ul><li>Insulin injection significantly increased nNOS phosphorylation at Ser 1416 in the NTS as compared to control. </li></ul><ul><li>LY294002 (PI3K inhibitor) significantly decreased nNOS phosphorylation in the NTS as compared to insulin group. </li></ul>In situ nNOS phosphorylation induced by insulin injection in the NTS <ul><li>All experimental group did not affect the level of nNOS in the NTS. </li></ul>Fig. 3 Western blot and immunohistochemical analysis of nNOS phosphorylation in the NTS after insulin injection
    17. 17. nNOS phosphorylation at ser 1416 induced by insulin injection in the NTS 1-3 1 Huang H. N. et al. 2004. Circulation. 110: 2476-2483 2 Yuan Z.R. et al. 2004. Brain research. 1008: 1-10 3 Vlahos C. J. et al. 1994. J Biol Chem. 269: 5241-5248 Results and discussion › › › Insulin-induced insulin receptor activity PI3K-Akt signaling pathway LY294002 nNOS phosphorylation at Ser 1416
    18. 18. <ul><li>nNOS can be phosphorylated by many kinases including PKA, PKC and CaMK II. </li></ul><ul><li>The phosphorylation of nNOS at Ser 1416 is essential for its activation. </li></ul><ul><li>The protein kinase responsible for Ser 1416 phosphorylation remains unclear. </li></ul>Results and discussion › › › Insulin activated Akt binds and phosphorylates nNOS at Ser 1416 1-3 PKA PKC CaMK II nNOS phosphorylation 1 Bredt D. S. et al. 1991. Nature. 351: 714-718; 2 Fulton D. et al. 1999. Nature. 399: 597-601; 3 Garcin E. D. et al. 2004. J Biol Chem. 279: 37918-37927 Abbreviation : PKA = protein kinase A; PKC = protein kinase C and CaMK II = Ca-calmodulin protein kinase II nNOS phosphorylation ??? ??? ???
    19. 19. Results and discussion › › › <ul><li>The results suggested that P-Akt 473 interact with P-nNOS 1416. </li></ul><ul><li>Akt did not affect the level of nNOS in the NTS. </li></ul>Insulin activated Akt binds and phosphorylates nNOS at Ser 1416 A B C Fig. 4 Insulin stimulate Akt kinase activity and nNOS function
    20. 20. Results and discussion › › › <ul><li>The results suggested that P-Akt 473 interact with P-nNOS 1416. </li></ul><ul><li>Akt phosphorylated nNOS at Ser 1416 in the time dependent manner from 0 - 120 min. </li></ul>Insulin activated Akt binds and phosphorylates nNOS at Ser 1416 A B C Fig. 4 Insulin stimulate Akt kinase activity and nNOS function
    21. 21. Results and discussion › › › <ul><li>The results suggested that P-Akt 473 interact with P-nNOS 1416. </li></ul><ul><li>Akt increased phosphorylation ratio in the time dependent manner from 0 - 120 min. </li></ul>p-nNOS/nNOS ratio Insulin activated Akt binds and phosphorylates nNOS at Ser 1416 A B C Fig. 4 Insulin stimulate Akt kinase activity and nNOS function
    22. 22. Summary › › › Insulin receptor (IR) Insulin nNOS activation is involved in insulin-mediated cardiovascular effect in the NTS P P P P IR substrate IR substrate PI3K P PI3K P PDK P PDK P Insulin-Pi3K-Akt pathway PIP 2 P P PIP 2 P P PIP 3 P P P PIP 3 P P P Ser 1416 P nNOS P nNOS-derived NO nNOS phosphorylation is induced by insulin injection in the NTS via PI3K-Akt signaling pathway 1 Akt P P Akt P P
    23. 23. Summary › › › nNOS activation is involved in insulin-mediated cardiovascular effect in the NTS nNOS is downstream factor in insulin-PI3K-Akt signaling pathway 2 Insulin receptor (IR) Insulin P P P P IR substrate IR substrate PI3K P PI3K P PDK P PDK P Insulin-Pi3K-Akt pathway PIP 2 P P PIP 2 P P PIP 3 P P P PIP 3 P P P Ser 1416 P nNOS P nNOS-derived NO Akt P P Akt P P
    24. 24. Summary › › › nNOS activation is involved in insulin-mediated cardiovascular effect in the NTS Hypotension Bradycardia ↓ Heart rate ↓ Arterial pressure The activation of nNOS-derived NO by insulin-PI3K-Akt signaling pathway participates in central cardiovascular regulation 3 Insulin receptor (IR) Insulin P P P P IR substrate IR substrate PI3K P PI3K P PDK P PDK P Insulin-Pi3K-Akt pathway PIP 2 P P PIP 2 P P PIP 3 P P P PIP 3 P P P Ser 1416 P nNOS P nNOS-derived NO Akt P P Akt P P
    25. 25. Summary › › › nNOS activation is involved in insulin-mediated cardiovascular effect in the NTS Hypertension Tachycardia ↑ Heart rate ↑ Arterial pressure Insulin receptor (IR) Insulin P P P P IR substrate IR substrate PI3K P PI3K P PDK P PDK P Insulin-Pi3K-Akt pathway PIP 2 P P PIP 2 P P PIP 3 P P P PIP 3 P P P Ser 1416 P nNOS P nNOS-derived NO Akt P P Akt P P The nNOS inhibitor and PI3K inhibitor reverse the cardiovascular responses of insulin 4 Vinyl-L-NIO 7-NI LY294002 LY294002
    26. 26. Thank You for Your Attention !!
    27. 27. Wistar-Kyoto (WKY) rats <ul><li>Coat color: white (albino) </li></ul><ul><li>Behavior: </li></ul><ul><li>Sensitive to the induction of ulcers by stress </li></ul><ul><li>Sensitive to salt ingestion </li></ul><ul><li>Kyoto School of Medicine </li></ul><ul><li>Control of spontaneously hypertensive (SHR) rats </li></ul><ul><li>Control of Attention deficit hyperactivity disorder (ADHD) model </li></ul>Backup › › › Wistar-Kyoto (WKY) rats Application Origin Feature
    28. 28. 1 Julian F.R.P. 1999. Exp physiol. 84: 815-833; 2 Andresen M. C. and Kunze D. L. 1994. Annu. Rev. Physiol . 56: 93-116 Backup › › › Nucleus tractus solitarius (NTS) 1, 2
    29. 29. Backup › › › Integrated neural pathway of NTS 1 1 Libby. Braunwald's Heart Disease, 8 th ed: 2007
    30. 30. 1 Klabunde R. E. Cardiovascular Physiology Concepts . Philadelphia: Lippincott Williams & Wilkins, 2005 Backup › › › The role of nucleus tractus solitarius (NTS) on cardiovascular activity 1 Visceral receptors Chemoreceptors Arterial baroreceptors Medulla oblongata Higher center Hypothalamus <ul><li>Vasoconstriction </li></ul><ul><li>Positive inotrophy </li></ul><ul><li>Positive chronotrophy </li></ul><ul><li>Positive dromotrophy </li></ul><ul><li>Negative inotrophy </li></ul><ul><li>Negative chronotrophy </li></ul><ul><li>Negative dromotrophy </li></ul>
    31. 31. 1 subsites.egaa-gym.dk/nr/biologi/roholt/Noter/note21a.html; 2 w ww.med.cmu.ac.th/dept/pharmaco/pharmacology/lesson01/03.htm Backup › › › The role of NTS on baroreceptor reflex 1, 2 Abbreviations <ul><li>VC: Vagal centers </li></ul><ul><li>VMC: Vasomotor center </li></ul>Glossopharyngeal nerve Vagus nerve
    32. 32. 1 Kevin J. T. 2009. Nature Reviews Immunology 9: 418-428. Backup › › › The role of NTS on inflammatory reflex 1 Abbreviations <ul><li>HPA axis: Hypotalamic-pituitary </li></ul><ul><li>-adrenal axis </li></ul><ul><li>NTS: Nucleus tractus solitarius </li></ul><ul><li>NA: Nucleus ambiguus </li></ul><ul><li>RVLM: Rostral ventrolateral medullary </li></ul>
    33. 33. 1 Ravinder K. et al. 2006. GI Motility online. doi:10.1038/gimo14 Backup › › › The role of NTS on swallowing 1 Mechanism Sensory signal Dorsal vagal nucleus Nucleus ambiguus Nucleus tractus solitarius (NTS) Lower esophageal sphincter (LES) Crural diaphragm Relaxation Contraction
    34. 34. Backup › › › Nitric oxide (NO) and NO synthase (NOS) 1, 2 Precursor Biosynthesis Distribution Chemical compound <ul><li>Free radical gas </li></ul><ul><li>L-arginine </li></ul><ul><li>Neuronal NOS (nNOS) </li></ul><ul><li>Epithelial NOS (eNOS) </li></ul><ul><li>Inducible NOS (iNOS) </li></ul><ul><li>Central nervous system (CNS) </li></ul><ul><li>Peripheral nervous system (PNS) </li></ul>1 Knowles R. G. and Moncada S. 1994. Biochem J . 298(pt. 2): 249-258 2 Moncada S. et al. 1991. Pharmacol Rev . 43: 109-142
    35. 35. Backup › › › 1 Richard Bruckdorfer. 2005. Molecular Aspects of Medicine 26:3-31 Structure and function of NOS isoforms 1 Oxygenase domain Reductase domain Abbreviations <ul><li>BH 4 : Tetrahydrobiopterin </li></ul><ul><li>FMN: Flavin mononucleotide </li></ul><ul><li>FAD: Flavin adenine dinucleotide </li></ul><ul><li>NADPH: Nicotine adenine dinucleotide phosphate </li></ul>
    36. 36. Backup › › › nNOS phosphorylation 1-4 1 Song T. et al. 2004. FEBS Letters . 570 :133–137 2 Rameau G. A. et al. 2004. J Biol Chem . 279(14): 14307-143014 3 Finocchietto P . et al. 2008. PLoS ONE. 3(3): e1749. doi:10.1371/journal.pone.0001749 4 Song T. et al. 2004. FEBS Letters . 570 :133–137 Enzyme Phosphorylate site of nNOS Effect on nNOS Ref. CaM-K I Ser 741 Inhibition 1 Cam-K II Ser 847 Inhibition 2 Akt Ser 1412 Activation 3 Akt Ser 1416 Activation 4
    37. 37. Backup › › › Comparison of 7-NI and vinyl-L-NIO NADPH FAD FMN Zn 2+ /BH 4 /Heme CaM COOH NH 2 L-arginine L-citrulline + Nitric oxide NADPH NADP + + H + Reductase domain (NOSred) Oxygenase domain (NOSox) PDZ nNOS Structure Vinyl-L-NIO 7-NI
    38. 38. 1 Herring N. and Paterson D. J. 2001. J Physiol . 535: 507-18 2 Paton J. F. R. et al. 2002. TRENDS in Neuroscience . 25(12): 626-631 Backup › › › The role of nNOS-derived NO on heart rate 1, 2 (via G i protein )
    39. 39. 1 Toda N. and Ayajiki K. 2010. Alcohol and Alcoholism . 45 (4): 347-355 Backup › › › The role of nNOS-derived NO on blood pressure 1
    40. 40. Abbreviation: PIP 2 = Phosphatidylinositol (PI) 4,5-bisphosphate, PIP 3 = PI 3,4,5-triphosphate Structure and classification BackUp › › › Phosphatidylinositol 3-kinase (PI3K) 1,2,3 PIP 2 PIP 3 PI3K - Lipase kinase 1 Ito K. et al. 2007. Perspectives in Pharmacology . 321 (1): 1-8 2 Oudit G. Y. and Penninger J. M. 2009. Cardiovascular Research . 82: 250–260 3 Bader A. G. et al. 2005. Nature Reviews Cancer . 5(12): 921-929 Catalytic subunit Regulatory adaptor subunit SH2/SH3 domain
    41. 41. Morpholine derivetive Reversible Competitive Cell-permeable <ul><li>LY294002 inhibits ATP binding site on the catalytic subunit of PI3K </li></ul>Backup › › › LY294002 (PI3K inhibitor) LY294002
    42. 42. Backup › › › 3-phosphoinositide dependent protein kinase (PDK) 1 1 Reuben J. et al. 2006. Nature. 441: 424-430 PDK1 PDK2 Akt Thr 308 Akt Ser 473 PDK
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