Oxide Synthase (NOS) is the enzyme that generates NO
This enzyme exists in three different forms (called
isoforms), NOS 1, NOS 2 and NOS 3. Each isoform synthesizes
NO but does so under different conditions. Often all
three isoforms will be found in the same cell but occasionally
one cell will contain only one of the isoforms. This
is important because many see or hear the term nitric
oxide and assume that it refers to all cells
under all conditions. Each of the three isoforms is
is the neural (or brain) isoform, sometimes referred
to as bNOS. It helps in synaptic transmission, the
processing of nervous information from nerve to nerve
across gaps between the nerves called synapses and
from peripheral nerves to the brain.
NOS2 is called inducible or iNOS. This enzyme
generates extraordinarily high concentrations of NO,
in part to kill bacteria. NOS2 (iNOS) takes several
hours to be mobilized and the response is due to an
injury or infectious process. NOS2 produced by macrophages
is responsible, in part, for their effects to repair
injury and to ward off infections. Extraordinarily
high concentrations of NO (100 to 1000 times normal)
are produced very locally by this isoform.
Unlike NOS1, which is part of normal neurotransmission,
there must be something very abnormal (a wound, tissue
damage, hypoxia, bacterial infection, etc.) to induce
The third isoform is ecNOS (or NOS3) which
stands for “endothelial cell” NOS. This isoform is
active at all times (it doesn’t need to be induced
as does iNOS) and is found in endothelial cells which
are the cells that line the inner surface of all blood
vessels and lymph ducts. EcNOS is activated by the
pulsatile flow of blood through vessels (the stretching
and relaxation of the blood vessel wall in response
to each beat of the heart).
This leads to a “shear stress” on the membrane
of the endothelial cells as the column of blood in
the vessel moves forward and then stops. This NO,
produced by ecNOS, maintains the diameter of blood
vessel so that perfusion of tissues (skin, muscle,
nerves, and bone) is maintained at optimal levels.
In addition, ecNOS mediated NO causes angiogenesis,
which is the growth of new blood vessels. This is
especially important in healing an ulcer or wound
on the skin.
initiates and maintains vasodilation through a cascade
of biological events that culminate in the relaxation
of smooth muscle cells that line arteries, veins, lymphatics.
While somewhat complex, the sequence of biological
events that are triggered by NO is described below:
1. NO gas released from nitrosothiols in hemoglobin
or from endothelial cells diffuses into smooth muscle
cells that line small blood vessels.
2.Once inside the smooth muscle cell, NO binds to an
enzyme, called guanylate cyclase (GC) and this binding
results in GC activation.
3. Activated GC is able to cleave two phosphate groups
from another compound called guanosine triphosphate
(GTP). This results in the formation of cyclic guanosine
monophosphate (cGMP) that is used to phosphorylate (Phosphorylation
is the addition of a phosphate group) proteins, including
the smooth muscle contractile protein called myosin.
phosphorylated, smooth muscle cell myosin relaxes, resulting
in dilation of the vessel that was originally exposed
vasodilation continues until a phosphatase enzyme dissociates
the phosphate from myosin (which may be delayed by Viagra).
Since vasodilation through NO only occurs when
there is GC able to bind NO, additional NO, is sequestered
for future use as a nitrosothiol, including those found
has both a direct and indirect influence on neurotransmission. NO, by affecting cGMP, allows phosphorylation of ion channels,
especially potassium channels necessary for normal transmission
of nerve signals. NO also increases blood flow. This
allows sufficient oxygen and glucose to be transported
to nerve cells, positively affecting ATP production
and, in turn, potassium/sodium homeostasis essential
for neurotransmission. Increases in blood flow may also
allow the oxygen dependent isoform, bNOS, to produce
addition to improving neurotransmission, NO functions
to reduce pain.
No reduces pain directly by increasing
cGMP (the mechanism by which opioids work), and indirectly
by increasing circulation to restore normal membrane
potential and reduce pressure on nerves due to localized
is important in the process wound healing and tissue
Oxide (NO) and its interrelationship with essential
growth factors is critically involved in the entire
continuum of events associated with wound repair.
NO is a powerful stimulator of cell division
(proliferation) and maturation, particularly formation
of appropriate cell receptors (differentiation).
NO is a necessary mediator of neovascularization,
i.e., the formation of new and eventually mature blood
vessels (angiogenesis) and lymph ducts to nourish the
NO increases the number of fibroblasts (fibroblastic
proliferation) and thereby enhances collagen formation
for the healing wound. Lastly, L-arginine and NO are
necessary for the proper cross-linking of collagen fibers
to one another, via proline, to minimize scarring and
maximize the tensile strength of healed tissue.
is often impaired in people with diabetes
Type I and Type II diabetic patients have a reduced
ability to generate NO from L-arginine, reflected in
part by direct measurements of plasma nitrate and nitrite
levels. Several factors influence nitric oxide production
part of normal metabolism of L-arginine small amounts
of a natural inhibitor of NOS are formed (asymmetrical
dimethyl arginine (ADMA). Normally, ADMA does not accumulate
in the blood because it is rapidly eliminated in the
urine through normal kidney function.
Reduced kidney function as part of aging (more
than 20% of all Americans over 65 have Type 2 diabetes)
or due to kidney dysfunction, which is accelerated by
diabetes, may prevent the elimination of the major NOS
inhibitor, ADMA, thereby limiting the production of
oxide synthase (NOS) from which NO is derived, is a
pH dependent enzyme.
It is active at slightly alkaline (basic) conditions
but is suppressed by acidotic conditions. In diabetes,
glycolysis and ketoacidosis force pH toward acid conditions
and this may account, in part, for the reduced production
Oxygen is necessary for the activity of NOS and therefore
NO. Circulation is notoriously impaired in diabetic
patients, which limits available NOS and NO.
people with diabetes often experience elevated glucose
levels. Some of this glucose becomes incorporated into
hemoglobin and is measured as glycosylated hemoglobin
(Hgb) or HgbA1C. Glycosylated hemoglobin binds NO in
the form of nitrosothiols very tightly so that any NO
that is formed cannot be easily released from RBC to
help maintain blood flow through smooth muscle cell
summary, acidosis, low oxygen, and/or accumulation of
ADMA are responsible for the decreased production of
NO is available is tightly bound to glycosylated hemoglobin
limiting its release and smooth muscle cells where NO
affects essential cellular functions.
NO deficiency may impair the health of people with diabetes
production and higher than normal binding, may be partly
responsible for the poor circulation in diabetic patients
and would be one of the reasons for their high propensity
to develop an ulcer.
Additionally, poor nitric oxide metabolism is
thought by some researchers to be the cause of peripheral
neuropathy, the nerve damage often observed in people
understanding the ways that NO can reduce pain, it is
easy to realize its significance in people with diabetes.
Impaired circulation is a typical consequence
of this disease. Disturbed membrane potential would
be anticipated thus decreasing the stimuli necessary
for nerve firing and perception of pain.
Additionally, this impaired circulation often
leads to swelling in the extremities, exerting pressure
on the nerves, which also causes pain.
Lastly, NO mediated increases in cGMP may be
impaired limiting its ability to directly reduce neuropathic
Tom Burke received his PhD in Physiology from University
of Houston, Post Doctoral Training at Duke Medical School,
He was an Associate Professor of Medicine and Physiology
at the University of Colorado Medical School. He has
authored more than 70 published scientific clinical
articles and has been a visiting scientist at the Mayo
Clinic, Yale University, University of Alabama, and
University of Florida. He is a recognized international
lecturer on cell injury and nephrology.