NitricoxideNeurotransmitter?

B.Sc2002

Oxygen and Nitrogen

:O

Oxygen 1s 2 2s 2 2p4

missing electron pair

. O:

Outer shell number = 12

Each atom now has share of 8 electrons
However molecular orbital considerations show that
two electrons are in unpaired orbitals with same spin
Oxygen is therefore a 'diradical'.

. N : O:
:

Nitric oxide
Outer shell number = 11

unpaired electron

N.

Electron number = 7
Outer shell number = 5
Three electrons needed for stable shell

Electron number = 8
Outer shell number = 6
Two electrons needed for stable shell

Dioxygen, molecular oxygen

Nitrogen 1s 2 2s 2 2p3

Dinitrogen, molecular nitrogen

:
:N : N :
:

Outer shell number = 10

Each atom now has share of 8 electrons
molecular orbital considerations show that
triple bond exists (one sigma and two pi bonding orbitals)
dinitrogen is therefore extremely stable

: N :: O .

Odd number of electrons;

free radical

NO is almost same size as dioxygen and can therefore fit in oxygen binding sites

EDRF:endotheliumderivedrelaxingfactor
Furchgottin1980showedthatAcetylcholinestimulated
relaxationofarterialsmoothmusclerequiredintact
endotheliuminthearteries
Palmer&Moncadainlate1980sshowedthatEDRFwas
nitricoxide,NO,synthesisedbyaspecificenzyme,Nitric
OxideSynthase(NOS)NOSusesarginineandoxygento
produceNOandcitrulline
DifferentformsofNOSexist.Constitutiveforms
andinducibleforms.NeuronalNOSisconstitutive,
macrophageNOSisinduced.

Mechanism of arginine oxidation by NOS

oxygen
1/2

O2

arginine

NH 2
+

N-hydroxy-arginine

NH

NH 2
Established
mechanism

NH

(CH 2 )

COOH

NH 2

N OH

C
NH

NH 2

nitric oxide

NH 2

NO

NH
(CH 2 )

COOH

citrulline

How does this occur

(CH 2 )
H

C
NH 2

N-hydroxy-arginine

NH 2

C
NH

(CH 2 )
H

N OH

COOH

C
NH 2

COOH

oxygen

NOS-a

arginine

superoxide?

NOS-b

N-hydroxy-arginine

nitric oxide

citrulline

NOsynthesisrequiresmolecularoxygen,
NADPH,andtetahydrobiopterin
TheintermediateNhydroxyargininecouldbea
storageformofNO.Superoxideanionmaybean
intermediateinNOsynthesis

BrainNOSispresentinCNSincludingspecificneuronsin
cerebellum,hippocampusandolfactorylobes;andinNANC(non
adrenergic,noncholinergic)nervesincludinginnervationto
gastrointestinaltract,pelvicorgansandtrachea.
VascularNOSispresentinplateletsandrenalmesangialcellsin
additiontoendothelium.
NOwasinitiallythoughttoisunstablebutisstableatthepartial
pressuresofoxygenfoundinthebody.Itismetabolisedtonitrite
andnitratewhichareexcretedinurineandprovideanindexofNO
biosynthesisinhumansreceivingalownitratediet.

HowdoesNOwork?
Diffusestocellsotherthantheonewhereitwassynthesised
BindswithsolubleguanylatecyclasetoformcGMP
cGMPactstosequesterintracellularcalciumandclosecalcium
channels
Dropinintracellularcalciumcausesrelaxation

HowisNOinactivated?
NOisoxidisedtonitriteandthennitrate
Nitrateexcretedinurine

FunctionsofNOinbody
Dilatesbloodvessels
Displacesoxygenfromoxyhaemoglobin
metabolicfactormediatingincreasedblood
flowtoexercisingmuscle
Appearstopreventhypoxia
Releasedfromorganicnitrateandnitrite
vasodilators
Actstorelaxbronchialsmoothmuscle

NOandhaemoglobin
Earlystudieswithfreehaemoglobin
showedthatNOconverteditirreversiblyto
methaemoglobin
Studieswithintacterythrocyteshave
suggestedthatNOcanbindreversiblyto
Hb,formingnitrosoHb
NitrosoHbmaybeconvertedbackto
oxyHbinlungs

WhatisphysiologicalroleofNO?
NOappearstoacttopreventhypoxiabycausing
vasodilation.
ItformsagoodmechanismtocounterhypoxiaBUT
Itneedsmolecularoxygentobesynthesised
NOisagasandcannotbestoredinthetissue
Couldtherebeastorageform?
ConceptthatNOisproducedduringperiodsofnormoxia,
storedasnitrosothiolornitrite,convertedbacktoNOin
hypoxicconditions
Maybehydroxyarginineisastorageform

AsensiblemodelofNOinthebodywouldexistifNO
couldbestoredinsomewayandformreversible
complexeswithhaemoglobin
Thenduringhypoxia
1) NOwouldbereleasedfromstoragetocauselocal
vasodilationandunloadingofoxygenfrom
haemoglobin,formingnitrosoHb
2) NitrosoHbiscarriedbacktolungsandisconverted
backtooxyhaemoglobin.ThereleasedNOrelaxes
thelungsandincreasedoxygenationofHb
Isnitritethestorageform?ThereMUSTbesomewayto
backconvertnitritetoNOasorganicvasodilators
(GTN,amylnitrite)areeffective!

WhatisroleofNOinbrain?
NOS(neuronal)isfoundinscattered
neuroneswithaxonsramifyingnear
cerebralbloodvessels
NOS(endothelial)isfoundinmany
astrocyteswithprocessesoncerebralblood
vessels
DoesNOcontrolcerebralbloodflow?

CerebralNOhypothesis:
NOScontrolsCBF.
DuringnormoxiaNOissynthesisedandblockadeof
thissynthesisreducesCBFandabilitytoautoregulate
FreshlymadeNOisusedtomaintaincalibreofcerebral
capillaries
DuringhypoxiaNOisreleasedfromstorageforms;thus
blockadeofNOSdoesnotaffectNOresponseto
hypoxia

Hypothesis2:RoleofNOinsupportingneuronalactivity
NOactstoincreaselocalbloodflowtoneuronesduring
increasedactivity.
NOthereforematchesperfusiontooxygendemand.
NOcontainingneuronesarestimulatedbyNMDAreceptors.
Thisallowscalciumentry,Ca/calmodulinactivatesNOSand
producesNOwhichdiffusestolocalbloodvesselsandcauses
dilation.CostoredwithGABAasGABAactstoreduce
excessactivity

Themainfeaturesofgenerallyaccepted
criteriaforaneurotransmitterareasfollows:
(1)Asystemforsynthesisoftheputativetransmittermustbe
present.
(2)Theremustbeastoreoftheputativetransmitterintheaxon
terminals.
(3)Theputativetransmittermustbereleasedbynerve
stimulation.
(4)Administrationoftheputativetransmittermustproducea
responsethatmimicsthatproducedbynervestimulation.
(5)Drugsthatmodifytheresponsestotheputativetransmitter
shouldhavecorrespondingeffectsontheresponsestonerve
stimulation.

CanNObeclassedasaneurotransmitter?
NerveterminalscontainNOS
NOsynthesisiscalciumdependent
NOisreleasedfromcertainautonomicneuronesafter
stimulation
NOcannotbeclassedasaclassicaltransmitterbecause(as
agas)itcannotbestoredinnerveterminals
ActionPotentialthatletsCaintopresynapticaxonmay
triggerNOsynthesis
NOisnotreleasedfromvesiclesbynerveactionbut
synthesisedaftereachAP

IsNOaretrogradetransmitter
BlockadeofNOSblocksLTPinhippocampalslices
NOmaybemadeinpostsynapticneuroneanddiffusebackto
presynapticneurone.Suggestedasamechanismofsynaptic
strengthening.Problemwiththisideaisthatnotallneurones
containNOS.

NO&Pain
ThereareconflictingreportsontheroleofNOinthetransmission
ofpaininthespinalcord.
NOSinhibitorshavebeenreportedtoreduceresponsestopain,but
sometimesincreaseresponses.
ThereisverygoodevidenceforincreasedNOsynthesisinthe
spinalcordinanimalswithchronicpain.
Thisincreasemayreflectmoreongoingactivityofneuronesin
chronicpainstatesratherthananincreaseinNOasapain
transmitter

ToxiceffectsofNO
NOonitsownisnottoxic,butitreactswithsuperoxideto
formperoxynitrite,atoxiccompound