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 › › ›

The insulin receptor in the central nervous system (CNS) 1,2

Insulin receptor

Generalization

CVSfunction

Location in CNS

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 role of insulin receptor in the CNS on cardiovascular function 1

1 Huang H. N. et al. 2004. Circulation. 110: 2476-2483.

Insulin receptor (IR)

Insulin

P

P

P

P

IR substrate IR substrate

PI3K PPI3KP

PDKP PDK PAktP P AktP P

NOS-derived NO ↓ Heart rate↓ Arterial pressure

Hypotension Bradycardia

Insulin-PI3K-Akt-NOS signaling pathway plays an important role in central cardiovascular regulation

Insulin-Pi3K-Akt pathway

Bradycardia is the resting heart rate of under 60 beats per minute

PIP2P P

PIP2P P

PIP3P PP

PIP3P PP


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)


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.

The insulin-mediated signaling in the NTS may play a significant role in the regulation of cardiovascular activity


The role of nNOS in NTS on cardiovascular function 1-6

1 Tseng C. J. et al. 1996. Hypertension. 27: 36-42; 2 Lo W. C. et al. 2004. Hypertension. 22: 36-423 Zhang K. et al. 1998. Brain Res. 786: 219-225; 4 Bredt D. S. et al. 1990. Nature. 347: 768-7705 Maeda M. et al. 1999. Neuroreport. 10: 1957-1960; 6 Ollerstam A. et al. 1997. J Clin Invest. 99: 2212-2218

High expression in NTSNeuronal nitric oxide synthase (nNOS)

Nitric oxide (NO) production

L-arginineL-arginine microinjection

nNOS inhibitor:7-nitroindazole (7-NI) nNOS antisense

oligonucleotide:

Inhibition

Blood pressure

The NO derived from nNOS in the NTS contributes to control cardiovascular effect.

Prominent depressor Bradycardic effecto Sympathetic outflow from brainstem

o Vasomotor toneo Blood pressure

Regulate

Cardiovascular function


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 regulation3

Experimental procedures › › ›

Experimental animals

Wistar-Kyoto (WKY) rats

Gender WeightMale 250-300 g

Hemodynamic measurement

300 mg/kg i.v.If necessary

1.0 g/kg i.p. Anesthesia - Urethane

NTS microinjectionDrug administration

• Insulin• PI3K inhibitor:

- LY294002• nNOS inhibitor:

- 7-nitroindrazole (7-NI) - Vinyl-L-NIO

•

Measurement

• Blood pressure: - Pressure transducer

- Polygraph• Heart rate:

- Tachograph amplifier


Western blot analysis 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.

Immunohistochemistry analysis

Immunohistochemical staining is used to determine nNOS phosphorylation after insulin

injection in the NTS of WKY rats.


Co-immunoprecipitation (Co-IP) assay

Co-IP assay is used to determine interaction between Akt and nNOS

In vitro kinase assay

In vitro kinase assay is used to determine Akt activity on nNOS phosphorylation


Statistical analysis

Experimental data

o Mean ± SEM.

Statistical difference

o P value < 0.05

Statistical significant

o Paired t-testo One-way ANOVAwith Dunnett ‘s test

Results and discussion › › ›

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.

The attenuation effect of 7-NI pretreatment lasted for more than

90 min. Pretreatment with 7-NI

significantly increased MBP and HR to compare the control.


Unilateral microinjection of insulin

The NTS of 8-week-old- WKY rats

Into

Prominent depressor

Produce

Bradycardic effect

Vinyl-L-NIO pretreatment

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.

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.

A

B

Pretreatment with vinyl-L-NIO significantly increased MBP and HR to

compare the control.


Inhibitory effect of the nNOS inhibitor on insulin-mediated cardiovascular effect 1-5

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

1 Huang H. N. et al. 2004. Circulation. 110: 2476-2483; 2 Tseng C. J. et al. 1996. Hypertension. 27: 36-42; 3 Lo W. C. et al. 2004. Hypertension. 22: 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.


Fig. 3 Western blot and immunohistochemical

analysis of nNOS phosphorylation in the

NTS after insulin injection

A B

C

Insulin injection significantly increased nNOS phosphorylation at Ser1416 in the NTS as compared to control.

LY294002 (PI3K inhibitor) significantly decreased nNOS phosphorylation in the NTS as compared to insulin group.

In situ nNOS phosphorylation induced by insulin injection in the NTS

All experimental group did not affect the level of nNOS in the NTS.


nNOS phosphorylation at ser 1416 induced by insulin injection in the NTS 1-3

Insulin-induced insulin receptor activity

PI3K-Akt signaling pathway

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

LY294002

nNOS phosphorylation at Ser 1416


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 can be phosphorylated by many kinases including PKA, PKC and CaMK II.

nNOS phosphorylation

The phosphorylation of nNOS at Ser 1416 is essential for its activation.

??? ???

???

The protein kinase responsible for Ser 1416 phosphorylation remains unclear.


Insulin activated Akt binds and phosphorylates nNOS at Ser 1416

A

B

C

Fig. 4 Insulin stimulate Akt kinase activity and nNOS

function

The results suggested that P-Akt 473 interact with P-nNOS 1416.

Akt did not affect the level of nNOS in the NTS.



A

B

C


function


Akt phosphorylated nNOS at Ser 1416 in the time dependent manner from 0 - 120 min.



A

B

C


function


Akt increased phosphorylation ratio in the time dependent manner from 0 - 120 min.

p-nNOS/nNOS ratio

Summary › › ›

nNOS activation is involved in insulin-mediated cardiovascular effect in the NTS

Insulin receptor (IR)Insulin

P

P

P

P


PI3K PPI3KP

PDKP PDK P


PIP2P P

PIP2P P

PIP3P PP

PIP3P PP

Ser 1416P

nNOS

P

nNOS-derived NO

nNOS phosphorylation is induced by insulin injection in the NTS via PI3K-Akt signaling pathway 1

AktP P AktP P

Summary › › ›


nNOS is downstream factor in insulin-PI3K-Akt signaling pathway2


P

P

P

P


PI3K PPI3KP

PDKP PDK P


PIP2P P

PIP2P P

PIP3P PP

PIP3P PP

Ser 1416P

nNOS

P

nNOS-derived NO

AktP P AktP P

Summary › › ›


Hypotension Bradycardia

↓ Heart rate↓ Arterial pressure

The activation of nNOS-derived NO by insulin-PI3K-Akt signaling pathway participates in central cardiovascular regulation

3


P

P

P

P


PI3K PPI3KP

PDKP PDK P


PIP2P P

PIP2P P

PIP3P PP

PIP3P PP

Ser 1416P

nNOS

P

nNOS-derived NO

AktP P AktP P

Summary › › ›


Hypertension Tachycardia

↑ Heart rate↑ Arterial pressure


P

P

P

P


PI3K PPI3KP

PDKP PDK P


PIP2P P

PIP2P P

PIP3P PP

PIP3P PP

Ser 1416P

nNOS

P

nNOS-derived NO

AktP P AktP P

The nNOS inhibitor and PI3K inhibitor reverse the cardiovascular responses of insulin4

Vinyl-L-NIO 7-NI

LY294002 LY294002

Thank You for Your Attention !!

Backup › › ›

Wistar-Kyoto (WKY) rats

Application

Origin Feature

Coat color: white (albino)

Behavior:

- Sensitive to the induction of ulcers by stress

- Sensitive to salt ingestion

Kyoto School of Medicine

Control of spontaneously hypertensive (SHR) rats

Control of Attention deficit hyperactivity disorder (ADHD) model

Backup › › ›

Nucleus tractus solitarius (NTS) 1, 2

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 › › ›

Integrated neural pathway of NTS 1

1 Libby. Braunwald's Heart Disease, 8th ed: 2007

Backup › › ›

The role of nucleus tractus solitarius (NTS) on cardiovascular activity 1

Visceral receptors

Chemoreceptors

Arterial baroreceptors

Medulla oblongata

Higher center Hypothalamus

1 Klabunde R. E. Cardiovascular Physiology Concepts. Philadelphia: Lippincott Williams & Wilkins, 2005

o Vasoconstriction

o Positive inotrophyo Positive chronotrophyo Positive dromotrophy

o Negative inotrophyo Negative chronotrophyo Negative dromotrophy

Backup › › ›

The role of NTS on baroreceptor reflex 1, 2

1 subsites.egaa-gym.dk/nr/biologi/roholt/Noter/note21a.html; 2 www.med.cmu.ac.th/dept/pharmaco/pharmacology/lesson01/03.htm

Abbreviations

VC: Vagal centers VMC: Vasomotor center

Glossopharyngeal nerveVagus nerve

Backup › › ›

The role of NTS on inflammatory reflex 1

Abbreviations

HPA axis: Hypotalamic-pituitary-adrenal axis

NTS: Nucleus tractus solitarius NA: Nucleus ambiguus

RVLM: Rostral ventrolateral medullary

1 Kevin J. T. 2009. Nature Reviews Immunology 9: 418-428.

Backup › › ›

The role of NTS on swallowing 1

Mechanism

1 Ravinder K. et al. 2006. GI Motility online. doi:10.1038/gimo14

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

Free radical gas L-arginine

Neuronal NOS (nNOS)

Epithelial NOS (eNOS)

Inducible NOS (iNOS)

Central nervous system (CNS)

Peripheral nervous system (PNS)

1 Knowles R. G. and Moncada S. 1994. Biochem J . 298(pt. 2): 249-2582 Moncada S. et al. 1991. Pharmacol Rev. 43: 109-142

Backup › › ›

1Richard Bruckdorfer. 2005. Molecular Aspects of Medicine 26:3-31

Structure and function of NOS isoforms 1

Oxygenase domain Reductase domain

Abbreviations

BH4: Tetrahydrobiopterin FMN: Flavin mononucleotide FAD: Flavin adenine dinucleotide NADPH: Nicotine adenine dinucleotide phosphate

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): 14307-1430143 Finocchietto P. et al. 2008. PLoS ONE. 3(3): e1749. doi:10.1371/journal.pone.00017494 Song T. et al. 2004. FEBS Letters. 570 :133–137

EnzymePhosphorylate site

of nNOSEffect on nNOS Ref.

CaM-K I Ser741 Inhibition 1

Cam-K II Ser847 Inhibition 2

Akt Ser1412 Activation 3

Akt Ser1416 Activation 4

Backup › › ›

Comparison of 7-NI and vinyl-L-NIO

NADPH FAD FMN Zn2+/BH4/HemeCaMCOOH NH2

L-arginine

L-citrulline+

Nitric oxide

NADPH

NADP+ + H+

Reductase domain (NOSred) Oxygenase domain (NOSox)

PDZ

nNOS Structure

Vinyl-L-NIO7-NI

Backup › › ›

The role of nNOS-derived NO on heart rate 1, 2

1 Herring N. and Paterson D. J. 2001. J Physiol. 535: 507-182 Paton J. F. R. et al. 2002. TRENDS in Neuroscience. 25(12): 626-631

(via Gi protein)

Backup › › ›

The role of nNOS-derived NO on blood pressure 1

1 Toda N. and Ayajiki K. 2010. Alcohol and Alcoholism. 45 (4): 347-355

BackUp › › ›

Phosphatidylinositol 3-kinase (PI3K)1,2,3

Abbreviation: PIP2 = Phosphatidylinositol (PI) 4,5-bisphosphate, PIP3 = PI 3,4,5-triphosphate

PIP2 PIP3

PI3K - Lipase kinase

1Ito K. et al. 2007. Perspectives in Pharmacology. 321 (1): 1-82Oudit G. Y. and Penninger J. M. 2009. Cardiovascular Research. 82: 250–2603Bader A. G. et al. 2005. Nature Reviews Cancer. 5(12): 921-929

Structure and classification

Catalytic subunit

Regulatory adaptor subunit

SH2/SH3 domain

Backup › › ›

LY294002 (PI3K inhibitor)

LY294002

Morpholine derivetive

ReversibleCompetitive

Cell-permeable

LY294002 inhibits ATP binding site on the catalytic subunit of PI3K

Backup › › ›

3-phosphoinositide dependent protein kinase (PDK) 1

1 Reuben J. et al. 2006. Nature. 441: 424-430

PDK1 PDK2

AktThr 308

Akt Ser 473

PDK

nnos activation is involved in insulin-mediated cv effects in the nts

Documents