nNOS activation is involved in insulin-mediated CV effects in the NTS

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

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

Introduction › › › 1 Huang H. N. et al. 2004. Circulation. 110: 2476-2483. Insulin receptor (IR) Insulin ,[object Object],[object Object],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

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)

Introduction › › › ,[object Object],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.

Introduction › › › High expression in NTS Neuronal nitric oxide synthase (nNOS) ,[object Object],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 ,[object Object],[object Object],[object Object],Inhibition Blood pressure Prominent depressor Bradycardic effect ,[object Object],[object Object],[object Object],Regulate Cardiovascular function

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

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

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 ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Measurement ,[object Object],[object Object],[object Object],[object Object],[object Object]

Experimental procedures › › › Western blot analysis ,[object Object],Immunohistochemistry analysis ,[object Object]

Experimental procedures › › › Co-immunoprecipitation (Co-IP) assay ,[object Object],In vitro kinase assay ,[object Object]

Statistical analysis Experimental procedures › › › Experimental data ,[object Object],Statistical difference ,[object Object],Statistical significant ,[object Object],[object Object],[object Object]

Results and discussion › › › ,[object Object],[object Object],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.

Results and discussion › › › ,[object Object],A B ,[object Object],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.

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

Results and discussion › › › A B C ,[object Object],[object Object],In situ nNOS phosphorylation induced by insulin injection in the NTS ,[object Object],Fig. 3 Western blot and immunohistochemical analysis of nNOS phosphorylation in the NTS after insulin injection

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

[object Object],[object Object],[object Object],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 ??? ??? ???

Results and discussion › › › ,[object Object],[object Object],Insulin activated Akt binds and phosphorylates nNOS at Ser 1416 A B C Fig. 4 Insulin stimulate Akt kinase activity and nNOS function

Results and discussion › › › ,[object Object],[object Object],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

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

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

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

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

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Wistar-Kyoto (WKY) rats ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Backup › › › Wistar-Kyoto (WKY) rats Application Origin Feature

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

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 › › › The role of nucleus tractus solitarius (NTS) on cardiovascular activity 1 Visceral receptors Chemoreceptors Arterial baroreceptors Medulla oblongata Higher center Hypothalamus ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

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 ,[object Object],[object Object],Glossopharyngeal nerve Vagus nerve

1 Kevin J. T. 2009. Nature Reviews Immunology 9: 418-428. Backup › › › The role of NTS on inflammatory reflex 1 Abbreviations ,[object Object],[object Object],[object Object],[object Object],[object Object]

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

Backup › › › Nitric oxide (NO) and NO synthase (NOS) 1, 2 Precursor Biosynthesis Distribution Chemical compound ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],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

Backup › › › 1 Richard Bruckdorfer. 2005. Molecular Aspects of Medicine 26:3-31 Structure and function of NOS isoforms 1 Oxygenase domain Reductase domain Abbreviations ,[object Object],[object Object],[object Object],[object Object]

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

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

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 )

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

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

Morpholine derivetive Reversible Competitive Cell-permeable ,[object Object],Backup › › › LY294002 (PI3K inhibitor) LY294002

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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