CARDIOVASCULAR JOURNAL OF AFRICA • Vol 23, No 4, May 2012
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AFRICA
three isoforms, namely neuronal NOS (nNOS), inducible NOS
(iNOS) and endothelial NOS (eNOS).
14,16,17
Physiologically,
eNOS and nNOS are constitutive, calcium-dependent enzymes
and continuously produce low levels of NO. On the other hand,
iNOS is calcium independent, its expression is provoked by
inflammatory cytokines, and it produces large amounts of NO,
about 1 000-fold more than eNOS or nNOS.
13
This can have
potentially harmful consequences as excess NO can react with
the free radical superoxide anion (O
2
–
), yielding a harmful and
highly reactive species, peroxynitrite.
13
All NOS isoforms require cofactors such as (6R)-5,6,7,8-
tetrahydrobiopterin (BH4), flavin adenine dinucleotide (FAD),
flavin mononucleotide (FMN), and iron protoporphyrin IX
(haem).
18
Of the three isoforms, it has been proposed that
eNOS is the major isoform responsible for NO production
under physiological conditions in the cardiovascular system and
endothelial cells in particular, leading to the classical signalling
mechanism in the underlying vascular smooth muscle cells
(VSMCs) and ultimately, relaxation
19
(Fig. 2).
Failure of the eNOS protein to dimerise, or the absence of
some of the cofactors mentioned above will lead to the enzyme
catalysing the formation of O
2
–
instead of NO, a mechanism
referred to as eNOS uncoupling
20
(Fig. 3). As will be discussed
later, eNOS uncoupling is an important mediator of ED during a
pathophysiological state.
Cardiovascular risk factors associated with
the development of ED
Type 1 diabetes mellitus and insulin resistance/type
2 diabetes mellitus
Both type 1 and type 2 diabetes are independent risk factors
for the development of accelerated atherosclerosis, IHD and
cardiovascular disease in general.
11
Similarly, type 1 diabetes
mellitus, insulin resistance and type 2 diabetes mellitus have
been shown to be strongly associated with the development of
ED.
21
In fact, the temporal progression from insulin resistance to
type 2 diabetes mellitus has been postulated to be mirrored by
the progression of ED to atherosclerosis.
22
ED observed in diabetes mellitus is primarily attributable to
(1) oxidative stress (increased O
2
–
generation due to upregulated
expression of NADPH oxidase), and (2) increased formation
of advanced glycation end-products (AGEs).
23,24
Aside from
scavenging NO, causing decreased NO bioavailability and
producing peroxynitrite, O
2
–
also modifies the activity and
regulation of eNOS, and promotes vascular smooth muscle cell
(VSMC) proliferation and inflammation.
24
Hyperglycaemia, as occurs in diabetes mellitus, results
in non-enzymatic glycation of intracellular and extracellular
proteins and lipids, which leads to the generation of AGEs. The
latter subsequently accumulate in the vascular wall and reduce
NO activity by quenching NO.
24,25
AGEs also bind to specific
surface receptors, called receptors for AGEs (RAGE), which are
expressed on cells such as monocytes, macrophages andVSMCs,
resulting in the amplification of an inflammatory response,
24,25
increased vascular permeability and oxidative stress.
25
Fig. 2. Synthesis of NO, downstream mechanisms and
physiological effects. NO is synthesised by eNOS in the
endothelial cells and diffuses into the underlying vascu-
lar smooth muscle cells (VSMCs), where it activates the
second messenger, cyclic guanosine monophosphate
(cGMP). Further downstream, signalling eventually leads
to VSMC relaxation and vasodilation. In addition, NO
regulates vascular homeostasis by anti-oxidation, anti-
inflammatory and anti-platelet aggregation effects.
Anti-inflammation:
leukocyte adhesion
and migration
Vascular lumen
Anti-oxidation
Anti-platelet aggregation
Endothelial cells
NO
NO
Vasodilation
VSMCs
eNOS
cGMP
Fig. 3. Coupled and uncoupled eNOS. (A) In the presence of sufficient levels of substrates and co-factors, and the
absence of harmful reactive species, eNOS monomers will form a dimerised, coupled enzyme and produce physi-
ological amounts of NO. (B) Decreased levels of the substrate, L-arginine and/or harmful effects exerted by increased
levels of ONOO
–
, cause failure of the enzyme to dimerise, leading to the uncoupling of eNOS and the production of
O
2
–
instead of NO.
HOOC–
HOOC–
NO
+
citrulline
NO
+
citrulline
O
2
+
l-arginine
O
2
+ ↓
l-arginine
O
2
+
l-arginine
O
2
+ ↓
l-arginine
Monomer
Monomer
N
2
H–
H
2
N–
Hommodimer:
eNOS
=
Coupled
eNOS
=
Uncoupled
e
–
e
–
e
–
e
–
e
–
e
–
NADP
+
NADP
+
FAD
FAD
FMN
FMN
BH
4
BH
3
Haem/Fe
2
+
Haem/Fe
2
+
Haem/Fe
2
+
Haem/Fe
2
+
BH
4
Zn
Zn
-COOH
-COOH
Monomer
Monomer
–NH
2
–NH
2
e
–
e
–
e
–
e
–
e
–
e
–
NADP
+
NADP
+
FAD
FAD
FMN
FMN
A
B
O
2
–
O
2
–
ONOO–
