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NITRIC OXIDE: FROM MENACE TO MARVEL OF THE DECADE

A  briefing  document  prepared  for  the  Royal  Society  and  Association  of  British  Science  Writers

Pearce  Wright

May  1996

Nitric  oxide:  From  menace  to  marvel  of  the  decade.

Summary  Research  papers  continue  to  flood  the  scientific  journals  with  insights  into  the  biological  activity  and  potential  clinical  uses  of  nitric  oxide  (NO):  a  gas  controlling  a  seemingly  limitless  range  of  functions  in  the  body.  Each  revelation  adds  to  nitric  oxide’s  already  lengthy  resume  in  controlling  the  circulation  of  the  blood,  and  regulating  activities  of  the  brain,  lungs,  liver,  kidneys,  stomach,  gut,  genitals  and other  organs.

The  molecule  governs  blood  pressure,  through  a  recently  recognised  process  that  contradicts  textbook  wisdom.  It  causes  penile  erections  by  dilating  blood  vessels,  and  controls  the  action  of  almost  every  orifice  from  swallowing  to  defecation.  The  immune  system  uses  nitric  oxide  in  fighting  viral,  bacterial  and  parasitic  infections,  and  tumours.  Nitric  oxide  transmits  messages  between  nerve  cells  and  is  associated  with  the  processes  of  learning,  memory,  sleeping,  feeling  pain,  and,  probably,  depression.  It  is  a  mediator  in  inflammation  and  rheumatism.

Clinically,  newborn  babies  with  breathing  problems  are  getting  relief  by  an  experimental  inhalation  treatment  developed  from  this  new  understanding.  A  novel  class  of  drugs  that  block  the  production  of  nitric  oxide  is  being  assessed  as  a  possible  treatment  for  septic  shock.  Drugs  that  liberate  or  enhance  the  action  of  nitric  oxide  may  be  useful  in  the  treatment  of  pulmonary  hypertension,  and  could  prevent  the  formation  of  blood  clots  and  counteract  impotence.  Animal  experiments  show  how  tomanipulate  nitric  oxide  production  to  stop  the  development  of  arthritis  and  kidney  disease.  In  the  longer  term,  drugs  that  alter  the  amount  or  activity  of  nitric  oxide  might  help  protect  the  brain  in  conditions  such  as  stroke,  Alzheimers  and  Parkinsons  disease.

Five  years  ago  the  Wellcome  Institute  for  the  History  of  Medicine  began  a  series  of  events  called  Witness  Seminars.  They  provide  a  forum  for  recording  for  the  archive,  first-hand  accounts  of  the  circumstances  leading  to  major  advances  in  contemporary  biomedical  science,  like  the  discovery  of  monoclonal  antibodies  and  unravelling  the  cause  of  thalassaemias,  a  group  of  inherited  blood  disorders  that  are  widely  spread across  the  Mediterranean,  Middle  East  and  Far  East,  and  cause  severe  anaemia.  They  capture  the  sort  of  reminiscences  and  perspectives  that  could  only  come  from  the  intimacy  of  an  autobiography  of  those  in  the  front  line  of  the  research.  A  similar  chance  to  savour  a  first-hand  narrative  of  an  area  of  research,  though  at  an  earlier  stage  of  science  in  the  making,  was  provided  by  Professor  Salvador  Moncada,  Dr  Patrick  Vallance,  Professor  John  Garthwaite  and  Professor  Eddy  Liew.  They  distilled  the  significance  of  a  welter  of  basic  and  clinical  research  by  scores  of  international  laboratories  into  how  nitric  oxide,  synthesised  from  the  amino  acid  Larginine,  literally  in  cells  from  head  to  foot,  can  keep  us  fit  and  healthy.

The  research  is  now  yielding  over  3  000  papers  a  year.  It  began  with  the  discovery  that  the  nitric  oxide  molecule  was  the  regulator  of  the  muscle  tone  of  blood  vessels.  While  the  finding  astonished  some  scientists  and  provoked  scepticism  in  others,  it  opened  a  floodgate.  The  potential  importance  of  the  torrent  of  research  results,  which  showed  nitric  oxide  governed  numerous  other  biological  processes,  received an  accolade  guaranteeing  wider  public  attention  when  nitric  oxide  was  chosen  as  the  cover  story  by  Science  for  its  “Molecule  of  the  Year”  in  1992,  and  was  wreathed  with  puns:  NO  sex;  NO  wonder;  NO  way.However,  it  couldn’t  match  the  vivid  account  of  “the  fascinating  adventure”  Salvador  Moncada  lives  when  recalling  the  research  that  unmasked  the  biological  role  of  nitric  oxide  and  the  subsequent  avalanche  of  work  it triggered.

He  was  a  senior  author  of  the  paper,  published  in  1987,  which  revealed  how  nitric  oxide  is  produced  to  control  the  relaxation  of  the  muscles  of  blood  vessels.  He  clearly  recalls  the  surprise  expressed  by  other  researchers  when  they  realised  that  such  a  crucial  activity,  with  profound  implications  for  understanding  the  cardiovascular  disease,  was  under  the  control  of  such  an  apparently  simple  molecule.

The  story  had  its  origins  seven  years  earlier.  In  effect,  the  gauntlet  was  thrown  down  by  R  F  Furchgott  and  J  V  Zawadski  who  showed  that  when  strips  of  blood  vessels,  nurtured  in  an  organ  bath,  were  chemically  stimulated,  the  muscles  relaxed.  If  the  inner  layer  of  cells  of  an  artery  or  vein,  the  endothelium,  was  absent,  the  smooth  muscles  of  the  blood  vessel  lost  their  capacity  to  make  the  vessel  expand.  This showed  that  a  previously  unrecognised  substance  must  exist  that  regulated  the  tone  of  the  smooth  muscles  of  blood  vessels.  They  referred  to  the  mystery  agent  as  endothelium  dependent  relaxing  factor,  EDRF.  Curiosity  provoked  several  laboratories  to  start  searching  among  the  body’s  complex  biomolecules  to  find  a  candidate  for  EDRF  among  the  array  of  amines,  peptides,  fatty  acids  and  other  chemicals.

Salvador  Moncada  and  his  colleagues  devised  two  experiments  to  test  whether  nitric  oxide  could  account  for  the  actions  of  EDRF.  The  first  was  to  determine  whether  nitric  oxide  was  released  by  endothelial  cells.  Equipment  developed  for  the  study  included  a  highly  sensitive,  miniaturised  version  of  an  instrument  used  in  the  car  industry  to  measure  nitric  oxide  in  the  exhaust  of  petrol  engines.  When  linked  to endothelial  cells,  repeated  measurements  demonstrated  that  nitric  oxide  was  indeed  the  relaxing  factor  released  by  these  cells.

The  second  experiment  compared  the  effects  of  natural  EDRF  on  muscle  tone  with  the  effects  produced  by  mimicking  the  relaxing  factor  with  “off  the  shelf”  nitric  oxide,  bought  from  the  British  Oxygen  Company,  the  specialist  that  made  industrial  gases  by  the  ton  rather  than  in  the  picogram  amounts  of  nitric  oxide  made  by  endothelial  cells.

Salvador  Moncada  recalled  that  nobody  had  the  remotest  suspicion  that  EDRF  would  turn  out  to  be  a  simple  molecule  like  nitric  oxide;  or  that  it  was  synthesised  not  just  in  the  blood  vessels,  but  as  an  essential  part  of  the  physiology  of  most  organs  and  tissues.

Previously,  nitric  oxide  was  regarded  as  an  environmental  pollutant  and  little  else:  at  best  a  chemically  reactive  nuisance,  at  worst  a  poison.  In  the  exhaust  fumes  of  cars  it  reacted  readily  with  oxygen  to  produce  smog,  increasing  the  risk  of  asthma.  When  discharged  into  the  atmosphere  from  power  station  chimneys  it  contributed  to  the  ecological  damage  from  acid  rain.  Consequently,  a  response  bordering  on  disbelief  greeted  the  discovery  that  cells  lining  the  walls  of  blood  vessels,  endothelial  cells,  intentionally  synthesised  nitric  oxide  as  a  muscle  relaxant.  The  molecule  is  shortlived,  and  a  constant  supply  is  generated  by  endothelial  cells  in  response  to  the  sheer  stress  of  the  blood  flow  on  the  artery  wall.  The  notion  that  such  a  noxious  little  molecule  should  also  hold  a  key  to  a  healthy  body  and  mind  was  counter-intuitive,
and  is  still  disconcerting  to  some  people.

Compared  with  the  complexity  of  the  hundreds  of  other  molecules  that  keep  us  ticking,  where  size  seems  to  equate  with  biological  relevance,  the  free  radical  form  of  nitric  oxide  that  constitutes  the  muscle  relaxing  factor  is  simplicity  itself:  just  one atom  of  oxygen  and  one  of  nitrogen.  However,  this  seemingly  uncomplicated  has  a  number  of  reactive  forms,  which  helps  explain  the  diversity  of  its  chemistry  and  the  range  of  biological  effects  they  stimulate.  Year  after  identifying  nitric  oxide  as  the  mystery  molecule,  Salvador  Moncada’s  group  reported  on  how  it  was  formed  in  endothelial  cells.  The  biochemistry  depends  on  the  action  of  one  of  a  family  of  enzymes,  nitric  oxide  synthases  or  NOS,  converting  the  amino  acid  L-arginine  to  L-citrulline  and  forming  nitric  oxide  in  the  process.

While  sceptics  still  thought  it  inconceivable  that  a  biochemical  pathway  for  producing  nitric  oxide  was  likely  to  exist  in  endothelial  cells,  they  were  batting  against  evolution.  It  transpires  that  the  ability  of  organisms  to  produce  nitric  oxide  is  an  ancient  one,  developed  long  before  mammals  emerged.  Indeed,  the  horseshoe  crab,  with  origins  going  back  500  million  years,  depends  on  the  L-arginine:  nitric  oxide  process  to prevent  its  blood  cells  from  aggregating.

The  early  research  overturned  some  basic  views  of  the  cardiovascular  system  whereby  blood  pressure  was  seen  as  resulting  from  a  constant  balancing  act  between  factors  influencing  the  constriction  and  the  dilation  of  vessels.  Changes  in  blood  pressure  and  blood  flow  happened  if  the  balance  was  tipped  too  far  one  way  or  the  other.  Now,  it  seems,  the  normal  cardiovascular  state  is  tilted  in  one  direction,  and the  dilation  of  vessels  is  sustained  by  a  steady  flow  of  nitric  oxide.  So  any  interruption  to  the  production  of  nitric  oxide  interferes  with  the  tone  of  the  smooth  muscle.

If  normality  means  that  blood  vessels  are  dilated  constantly,  then  the  prevailing  perception  of  blood  pressure  and  the  approach  to  managing  hypertension  may  need  a  major  overhaul.  From  the  new  point  of  view,  raised  blood  pressure  may  be  as  much  a  problem  of  lack  of  dilator  tone  as  it  may  be  due  to  constriction  caused  by  some  unknown  factor.

The  new  insight  also  explains  at  last  the  discovery  made  more  than  100  years  ago  that  a  group  of  drugs,  based  on  amylnitrite  and  nitroglycerine,  could  stop  a  painful  attack  of  angina:  the  chest  pain  now  known  to  be  caused  when  the  heart  muscle  is  short  of  oxygen.  By  chance,  the  Victorian  physicians  had  worked  out  that  although  it  was  too  dangerous  to  give  people  nitric  oxide  directly,  substances  that  released  it  slowly  could  relieve  hypertension.  The  helpful  effect  on  blood  pressure  of  a  whiff  of  amylnitrite  vapour  was  recognised  as  long  ago  as  1867,  and  Conan  Doyle  credited  Sherlock  Holmes  with  that  knowledge  in  The  Case  of  the  Resident  Patient.

The  poisonous  effects  of  nitric  oxide  were  well  known.  It  was  easy  enough  to  make  in  the  laboratory  by  adding  copper  turnings  to  concentrated  nitric  acid,  and  collecting  the  colourless  gas  over  water.  It  nearly  killed  Sir  Humphry  Davy,  in  1800,  when  he  experimented  with  breathing  it  in.

In  World  War  I,  doctors  noticed  that  workers  packing  shells  with  nitroglycerine  in ammunition  factories  had  very  low  blood  pressures.  The  observation  led  to  the development  of  a  pill  containing  nitroglycerine,  that  remains  effective  as  an emergency  vasodilator  when  popped  under  the  tongue  to  give  rapid  relief  for  angina. Although  effective,  the  precise  way  in  which  the  nitric  oxide  is  released  from  nitroglycerine  to  imitate  the  L-arginine  source  of  the  molecule,  is  still  a  bit  of  a  mystery.  Nitric  oxide  is  changed  rapidly  once  in  the  bloodstream  because  it  is  highly attracted  to  the  iron  in  haemoglobin.

As  research  into  nitric  oxide  gathered  momentum  nine  years  ago,  investigations spread  way  beyond  the  effect  of  the  endothelial  derived  molecule.  Three  forms  of  the NOS  enzyme  were  found:  one  in  the  endothelium,  one  in  the  brain  and  one  in  the immune  system.  Moreover,  the  biochemists  were  intrigued  to  discover  that  these enzymes  were  unusual  in  that  they  controlled  a  two  stage  reaction  that  would  usually take  two  enzymes.  More  recently,  molecular  biologists  have  homed  in  on  the different  genes  that  encode  for  the  endothelial,  neuronal  and  immune  system  nitric oxide  synthase  enzymes:  the  genes  are  on  chromosomes  12,  7  and  17  respectively. From  a  clinical  perspective,  Patrick  Vallance  outlined  the  far-reaching  implications  of nitric  oxide  for  treating  various  forms  of  cardiovascular  diseases,  which  account  for almost  half  the  deaths  in  Britain.  A  vast  range  of  research  is  aimed  at  translating  the laboratory  and  animal  findings  about  nitric  oxide  for  the  benefit  of  the  cardiovascular and  heart  disease  sufferers.

His  group  used  healthy  volunteers  to  test  whether  nitric  oxide  acted  on  the  human cardiovascular  system  in  an  identical  way  to  that  predicted  from  in  vitro  and  animal work  in  the  laboratory.  The  study  entailed  what  has  become  a  new  class  of  drug based  on  N-monomethyl-arginine  (L-NMMA  –  an  inhibitor  of  the  NOS  enzyme).  This drug  is  one  of  a  class  of  inhibitors  that  can  be  used  to  explore  what  happens  when nitric  oxide  production  is  blocked.  In  the  experiment,  one  forearm  of  a  volunteer  was injected  with  L-NMMA.  The  blood  flow  was  then  compared  with  that  of  the  other  arm.As  L-NMMA  was  infused  gradually,  blood  flow  decreased  to  a  half  of  that  in  the control  arm.

Complications  caused  by  problems  with  endothelial  production  of  nitric  oxide  arise from  a  variety  of  causes:  the  fragile  layer  of  endothelial  cells  are  prone  to  damage, from  among  other  things,  high  blood  pressure,  high  sugar  in  diabetes,  furring  up with  cholesterol  and  other  lipids,  and  the  effect  of  smoking.

In  cases  involving  a  risk  of  heart  attack,  attention  focuses  on  nitric  oxide  deficiency, and  the  dangers  when  its  release  is  blocked  from  a  diseased  coronary  artery  so  that vasodilator  tone  needed  to  regulate  the  flow  of  blood  is  lost.  Other  diseases  can  also be  treated  by  increasing  the  supply  of  nitric  oxide  to  blood  vessels.  A  trace  of  nitric -xide  gas  (25  parts  per  million)  inhaled  by  patients  with  pulmonary  hypertension  can relieve  lung  congestion.  In  a  treatment  for  newborn  babies,  breathing  problems  are being  helped  by  inhalation  of  nitric  oxide  that  relaxes  constricted  blood  vessels  and dilates  the  lung’s  blood  vessels.

Generally,  low  blood  pressure  is  not  a  problem  in  healthy  people.  Occasionally,  it  is  a symptom  of  serious  condition;  and  an  acute  drop  can  signal  that  an  overwhelming infection  has  caused  septic  shock  and  severe  tissue  damage.  The  trouble  lies  in  a vicious  cycle.  Septic  shock  depresses  blood  pressure.  The  loss  of  blood  flow  can cause  tissue  damage,  and  that  in  turn  increases  the  production  of  nitric  oxide, leading  to  a  further  fall  in  blood  pressure.  Low  blood  pressure  induced  by  septic shock  has  been  restored  with  a  treatment  that  included  the  use  of  an  NOS  inhibitor. The  remarkable  role  nitric  oxide  plays  as  a  messenger  between  nerve  cells  was revealed  by  John  Garthwaite  and  colleagues,  at  Liverpool  University,  who  first identified  it  in  the  brain.  Subsequent  measurements  have  shown  that  the  brain contained  more  of  the  nitric  oxide  synthase  enzyme  than  any  other  organ.

When  research  into  the  possible  role  of  nitric  oxide  in  the  central  nervous  system began,  the  brain  was  the  least  expected  organ  in  which  to  find  the  molecule.  It transpired  that  the  nitric  oxide  synthase  enzyme  was  not  confined  to  small  areas,  but was  distributed  throughout  the  brain:  suggesting  an  involvement  in  almost  every brain  function.  It  turns  out  that  because  the  molecule  is  so  small,  it  is  a  physically convenient  messenger  that  diffuses  into  and  out  of  cells  easily;  and  it  is  now  a  prime candidate  for  the  much  searched  for  “retrograde  messenger”  that  is  the  basis  ofmemory.

Animal  studies  suggest  that  an  effector  cell  in  the  brain  which  releases  the  chemical messenger  glutamate  can  stimulate  the  receptor  cell  with  this  chemical  to  release nitric  oxide.  If  it  is  strongly  stimulated,  the  receptor  sends  back  a  nitric  oxide molecule  to  tell  the  sender  that  the  message  has  been  received,  and  programmes  it to  send  an  even  stronger  signal  next  time  –  the  phenomenon  of  long  term potentiation  that  is  thought  to  be  linked  to  memory  formation. Nitric  oxide  produced  in  the  brain  by  the  neuronal  form  of  the  nitric  oxide  synthase acts  as  a  chemical  messenger  at  the  synapses.  The  in-vitro  and  animal  experiments showing  the  role  of  neuronal  nitric  oxide  in  memory  has  opened  a  new  approach  to studies  of  Alzheimers,  Parkinsons  and  other  neurological  disorders.

Too  much  nitric  oxide,  on  the  other  hand,  may  be  responsible  for  killing  brain  cells  in conditions  such  as  stroke.

In  yet  another  role,  nitric  oxide  helps  the  immune  system’s  macrophages,  the  cells  in the  body  that  are  activated  in  injury  and  illness  to  get  rid  of  debris,  when  they  seek out  invading  bacteria  or  cancer  cells  to  destroy.  As  the  “bin  men”  of  the  immune system,  the  macrophages  use  the  cytotoxic  properties  of  nitric  oxide  to  sweep  up parasites,  bacteria  and  other  potentially  infectious  rubbish.

Eddy  Liew’s  work  on  the  role  of  nitric  oxide  in  the  immune  system  shows  the molecule  will  destroy  many  organisms  that  are  difficult  to  kill  by  other  means.  And his  group  has  demonstrated  the  susceptibility  to  nitric  oxide  of  the  vicious  parasite that  causes  Leishmaniasis,  a  group  of  potentially  fatal  tropical  diseases  caused  byparasites  transmitted  by  the  bite  of  sandflies.

Treatment  with  the  inhibitor  L-NMMA  also  appears  to  block  inflammation  in  the  body associated  with  the  development  of  arthritis  and  kidney  disease.  Moreover,  mice genetically  engineered  not  to  generate  nitric  oxide  during  the  immune  responses display  a  reduced  inflammatory  response.  If  research  now  underway  confirms  the scientists’  hypotheses,  the  advances  could  lead  rapidly  to  a  new  class  of  drugs  to control  inflammatory  diseases  that  are  classified  as  auto-immune  diseases  because the  affected  tissue  is  under  continual  attack  by  its  own  immune  system. Research  on  nitric  oxide,  its  physiological  and  pathological  roles  and  clinical  potential is  currently  one  of  the  most  exciting  sources  of  knowledge  in  biology.