serve as a carrier and preserver of NO bioactivity in the circulation. It has been shown
that NO can react with Hb in two rather different ways: binding to haem-Fe2+ in a
process comparable to the binding of O2; and forming an adduct with surface-exposed
Cys93 p side chains.
According to the model described by Stamler105;110;113- also known as S-
nitrosohaemoglobin hypothesis- NO is first captured by the Fe2+ at the haem and then transferred to the sulfhydryl (-SH)group of the P-chain cysteine-93 (pCys93) residues
to form SNO-Hb. The haem iron preferentially binds NO when in the T (deoxy-)
conformation. The NO is transferred to pCys93 when Hb is in the R (oxy-)
conformation. Then when R changes again to the T conformation to deliver O2 to the
tissues, NO is also released in an allosterically dependant manner114 and transferred
(via trans-nitrosation reactions) to the sulfhydryl groups of small sulfhydryl molecules
(X-SH) such as glutathione to form X-S-NO. X-S-NOs, while having the same
vasodilator properties as free NO, are resistant to scavenging by haemoglobin. Inside
the RBCs, there is equilibrium between NO bound to the thiol of glutathione and
reactive thiols (cysB93) of haemoglobin on the one hand, and NO bound to the thiols
low oxygen tensions, NO is transferred from SNO-Hb in the cytosol to the membrane
to form membrane-SNO which can induce vasodilation105.
The net effect is the conversion of unstable free NO to relatively stable X-S-NO
which is oxygen-sensitive and can release NO in low oxygen conditions to relax the
vascular smooth muscle cells and dilate the arteries (hypoxic vasodilation).
Stamler and colleagues have reported several observations that support the S-
nitrosohaemoglobin hypothesis that have yet to be countered convincingly by other groups. First and foremost, they demonstrated the very existence of S-
nitrosohaemoglobin in the circulation113 and that SNO-Hb can be produced in vitro
when Fe(II)NO species are subjected to mild oxidation115. The same group reported a
significant arterio-venous gradient of SNO-Hb suggesting its cyclic metabolism in the
circulation116;117.
Challenges to the S-nitrosohaemoglobin hypothesis-Many aspects of the S-
nitrosohaemoglobin hypothesis have been questioned by other groups. Other laboratories have not been able to measure the micromolar concentrations and artery-
to-vein gradient of SNO-Hb reported by the Stamler group62’118,119. (A previous paper
from our laboratory reported increased levels of SNO-Hb with oxygenation across the
pulmonary circulation in patients with congestive heart failure but not in healthy
controls120.) Nor have they been able to reproduce the observation of preferential
binding of NO on the deoxyhaems of R-state (oxygenated) haemoglobin (i.e.,
allosterically controlled association kinetics) or even the oxygenation dependent
transfer of the NO from the 6-chain haem to the cysteine 93 and the deoxygenation-
dependent transfer of NO from the cysteine 93 back to the haem (i.e., cycling)84;121.
N itro sy lh a em o g lo b in (HbNO)
Most o f the NO w hich enters the erythrocytes will react with oxyhaem oglobin to form nitrate and m ethaem oglobin. N onetheless, some NO will m eet non-oxygenated
9 -4- 7 7 * 1 7 9 •
haem oglobin and nitrosylate the Fe" to a fairly stable HbNO adduct ’ (in vivo h a lf life ~ 40 m in u tes123’124). HbNO is also produced from the reaction betw een the continuous flux o f plasm a nitrite into the erythrocytes and non-oxygenated haem oglobin28. Therefore, HbNO is a co-index o f NO and nitrite uptake by erythrocytes at any given haem oglobin oxygen saturation.
H bN O is eventually oxidised by O2 to m ethaem oglobin and nitrate in three steps as
shown by H erold and R o ck 125:
Hb (F e ll2+) NO + 02 «-► Hb (Fe2+) + NO + 02 <-► Hb (FeII2+) 0 2 + NO —> M etH b +
N O3" (Equation 4)
NO u q OM*
C j f e > ♦ o , C p £ > ♦ O* ♦ NO £ = ? O f " • ♦ N O ' ♦ *°:>
HD Nb Hb Hb
F ig u re 1.6: T h ree-step d eg r a d a tio n o f H b N O to n itrate and m etH b . (F ro m H ero ld and R ock 2 0 0 5 125)
HbNO is one o f the m any m olecules proposed to serve as potential preservers o f NO bioactivity in the circulation. By binding to haem oglobin, NO avoids degradation to nitrate. H ow ever, HbN O does not show vasodilator properties in v iv o122,126 nor is
there any evidence that it can dissociate efficiently to deliver NO to tissues directly.
HbN O has a characteristic EPR (electron param agnetic resonance) spectrum . EPR has been w idely used to m easure changes in HbNO levels follow ing addition o f NO or its m etabolites to blood, in both in vitro and in vivo studies. However, because the level
of HbNO present in both arterial and venous samples at base line are below the
detectable level by EPR (<0.5 pM), EPR studies have not been able to show any
measurable A-V gradient in HbNO levels121,123. Our triiodide studies show higher
HbNO levels in venous blood (see CHAPTER FIVE).
RBC nitrite
Plasma nitrite constantly enters the RBCs72. Inside the erythrocytes, nitrite can either
react with oxyhaemoglobin to form methaemoglobin and nitrate (Equation 3); or with
deoxygenated haemoglobin to form methaemoglobin and nitrosylhaemoglobin
(HbNO)76;78 (Equation 4):
Hb02 + 2N02" —> MetHb + 2N03' (Equation 5)
4Hb + 2N02" + 2H+ -► 2MetHb + 2HbNO + 2H20 (Equation 6)
Nitrite concentrations in RBCs were long thought to be negligible because of the rapid
kinetics of the above reactions. However, recent studies by Kelm/Gladwin/Feelisch
teams have shown substantial levels of intraerythrocytic nitrite (200-500nM)68,127.
Virtually all nitrite was located in the cytosol as bound to proteins. They also found an
artery-to-vein gradient of RBC nitrite across the human forearm circulation, consistent
with their previous finding of artery-to vein-gradient in plasma.
Nitrated lipids (Nitrolipids)
Unsaturated fatty acids are nitrated endogenously to produce nitrated lipids128. Recent
studies have shown that these nitrated lipids may directly or through transnitrosation
reactions act as NO-releasing agents129. Nitrolipids relax rat aortic rings in a
concentration-dependent manner while releasing nitric oxide129. Nitrolinoleate, a
synthetic nitrated lipid, has been shown to inhibit platelet aggregation, probably
through a non-NO dependant mechanism130.