Chapter 8 Pharmacology of nitric oxide

8.1 Physiological significance of nitric oxide (NO)

Powerful vasodilatorNO-releasing drugs are used in the treatment of cardiovascular disease
Inflammatory mediatorinhibition of NO synthesis is of interest as a therapeutic strategy in
infection and chronic inflammation
Neurotransmittersignaling in the CNS and the autonomic nervous system
Nitric oxide differs in many ways from other signaling molecules, and because of its unusual nature
was discovered relatively late. Its first physiological function to be discovered was the relaxation of
blood vessels. While this function remains the focus of its application in pharmacotherapy, a range
of other effects with at least potential applications in clinical practice were characterized
subsequently.
The three listed physiological functions correspond to three different isoforms of nitric oxide
synthase, the enzyme that produces NO (see slide 8.3).
8.2 Identification of endothelium-derived relaxing factor as nitric oxide

Cholinergic and adrenergic nerve endings in a blood vessel wall

The endothelium is required for vascular relaxation in response to acetylcholine

Vasorelaxation mediated by nitric oxide is controlled by the autonomic nervous system. The
physiological experiments that uncovered the role of the endothelium, and subsequently of NO, in
vasorelaxation are simple yet ingenious. In order to appreciate them, we need a little more anatomy.
8.2.1Cholinergic and adrenergic nerve endings in a blood vessel wall

We had seen earlier that both the sympathetic and the parasympathetic nervous system innervate
blood vessel walls and regulate their contraction and wall tension (slide 6.13.1). The adrenergic
nerve endings of the sympathetic nerve fibers are found in the muscular layer of the vessel walls. In
contrast, cholinergic nerve endings are found both in the muscular and the endothelial layers.
The wall tension of the blood vessel is sustained by the muscular layer, not the endothelium;
therefore, the cholinergic innervation of the endothelium is surprising. As it turns out, however, it is
needed for the vasorelaxation and vasodilation induced by the parasympathetic system.
8.2.2 The endothelium is required for vascular relaxation in response to acetylcholine

This slide illustrates the experiment that led to the discovery of nitric oxide-mediated
vasorelaxation.
Contraction and relaxation of the vascular smooth muscle cells in response to norepinephrine and
acetylcholine were studied with aortic strips. In this technique, slices are cut from an animals aorta
and then opened with a radial incision. The endothelium can be left in place or peeled away in order
to study its effect on the muscular layer. The resulting intact or denuded aortic strips were mounted
between two distending hooks to measure their contractile force.
Application of norepinephrine induced contraction in strips with or without attached endothelium,
as would be expected from the innervation pattern of the sympathetic nerve fibers. In contrast,
norepinephrine-induced contraction could be reversed by acetylcholine only if the strip retained the
endothelium (top) but not when the endothelium had been peeled away.
Interestingly, relaxation of a denuded muscular strip could be restored if the endothelial side of an
intact strip was strapped onto it (not illustrated). This observation showed that, in response to
cholinergic stimulation, the endothelium releases a diffusible substance that enters the muscular
layer and induces its relaxation. This endothelium-derived relaxing factor was subsequently
isolated and identified as NO.

8.3 The nitric oxide synthase reaction

Activation of NOS by calcium and calmodulin

Nitric oxide is synthesized intracellularly by nitric oxide synthase (NOS). This reaction is rather
complex and involves two successive monooxygenase steps. In the first step, arginine is converted
to N-hydroxyarginine (NOHA), which is cleaved in the second step to NO and citrulline.
NOS occurs in several variations. Endothelial NOS (eNOS) and neuronal NOS (nNOS) are found in
the eponymous cell types. Inducible NOS (iNOS) is found mainly in inflammatory cells. All three
enzymes are homologous and perform the same reaction, but they differ in their regulatory
properties.
8.3.1Activation of NOS by calcium and calmodulin
Nitric oxide synthase exists as a dimer. Each subunit contains a reductase domain and an oxidase
domain. The reductase domain accepts electrons from NADPH and hands them over to the oxidase
domain of the other subunit.
The reductase and oxidase domains are loosely connected by flexible hinges that, when extended,

prevent the flow of electrons between them. The two domains are brought into close, productive
interaction when calmodulin (CaM) binds to the hinges and changes their conformations. In the
case of eNOS and nNOS, CaM binding only happens if the cytosolic level of Ca++ is elevated,
which occurs only transiently through action potentials or through activation of GPCRs (for
example the muscarinic ones in endothelial cells).
In contrast, inducible NOS binds calmodulin avidly and therefore is active even at the low resting
level of Ca++. Its activity is regulated not by short-term calcium signals but instead by
transcriptional induction.

8.4 NO activates soluble guanylate cyclase (sGC)

Signaling effects of cGMP

NO-induced relaxation of smooth muscle cells is mediated by cGK

Between the last slide and this one, we have skipped an important step, namely the diffusion of NO
from the cell of origin, for example an endothelial cell, to another one, such as a vascular smooth
muscle cell. Like other small gas molecules, nitric oxide passes through cell membranes with ease,
which enables it to directly and rapidly interact with intracellular targets.
Within its target cell, NO binds to the heme group of soluble guanylate cyclase (sGC), dislodging a
strategic histidine residue. This causes a conformational change that activates the enzyme, which
then begins to make cyclic GMP (cGMP). The production of cGMP as a second messenger is the
most important signal downstream of NO.
8.4.1Signaling effects of cGMP

Like cAMP, cGMP targets multiple effector molecules inside the cell. Activation of cGMPdependent protein kinases (cGK) is the most important single mechanism. Phosphodiesterase 5
(PDE) is activated, too, reducing the levels of both cAMP and cGMP. Actuation of cyclic
nucleotide-gated cation channels affects the membrane potential and the cellular calcium level.
Membrane-bound receptor or particulate guanylate cyclases (pGC) provide an alternate means of
cGMP production that is independent of NO but instead is controlled by several peptide hormones.
8.4.2NO-induced relaxation of smooth muscle cells is mediated by cGK

The relaxation of smooth muscle that occurs downstream of endothelial NO release is mediated by
cGMP-dependent protein kinase. This kinase phosphorylates and thereby activates myosin light
chain phosphatase. The phosphatase dephosphorylates myosin, which interrupts interaction of
myosin with actin and induces relaxation.
cGK also phosporylates a regulatory subunit of the IP3 receptor channel. As we have seen before
(slide 5.3.2), this channel mediates the outflow of Ca++ from the ER to the cytosol.
Phosphorylation of the IP3 channel reduces Ca++ flow and therefore the calmodulin-dependent
activation of myosin light chain kinase (MLCK).
It is worth noting that the effects of NO cut in below the GPCRs that mediate the effects of major
vasoconstrictors such as angiotensin and norepinephrine. Therefore, NO-releasing drugs offer a
means to interrupt the out-of-control signaling by such mediators that occurs in hemodynamic
shock or hypertensive crisis.
8.5 S-nitrosylation of cysteine residues in proteins by NO

Transfer of nitrosyl groups between proteins by glutathione

Identification of cysteines affected by S-nitrosylation: The biotin switch
Identification of proteins subject to nNOS-dependent S-nitrosylation

In addition to the cGMP-mediated signaling effects, nitric oxide can also influence protein function
through reacting covalently with them. The reaction involves single cysteine residues in proteins,
which become S-nitrosylated. The reaction also requires molecular oxygen, which is converted to
superoxide. While the latter has been observed in vitro to react with a second molecule of NO to
form peroxynitrite, it seems likely that in vivo most of it would be scavenged by superoxide
dismutase.
One might expect this modification to be too indiscriminate to be of use in selective and directed
control of cell physiology. However, it has been demonstrated experimentally that S-nitrosylation is
indeed quite selective in vivo.
8.5.1Transfer of nitrosyl groups between proteins by glutathione

One mechanism that may contribute to the selectivity of protein-S-nitrosylation in vivo is the
transfer of nitrosyl groups between proteins, which is mediated by glutathione. In this way, the
6

nitrosyl groups can travel around the cell before settling down on the proteins with the
thermodynamically most favorable cysteine residues.
Since glutathione is the most abundant thiol in the cell, this also means that nitrosylated glutathione
functions as a reservoir of SN=O groups in the cell. In fact, in many in vitro studies on the
regulatory effects of protein-S-nitrosylation, nitrosylated glutathione was used as a source of
nitrosyl group instead of free NO for simplicity.
8.5.2 Identification of cysteines affected by S-nitrosylation: The biotin switch

To understand the regulatory effects of protein-S-nitrosylation, it is important to identify the

proteins, and the specific cysteines within them, that actually become S-nitrosylated in vivo upon
NOS activation. The biotin switch method is an experimental protocol that permits the identification
of these cysteines by selectively attaching biotin to them. This procedure involves the following
steps:
After NOS has been activated and protein-S-nitrosylation has run its course, the remaining free SH
groups are converted to disulfides using S-methyl-methanethiosulfonate.
Ascorbic acid is used to selectively reduce SNO groups, while leaving native or synthetic disulfide
bonds intact.
The reduced SH groups are labeled with a biotinylation reagent, via disulfide formation (as shown
here) or other coupling chemistries.
After this procedure, biotin is attached specifically to those cysteine residues that had been
nitrosylated before. The biotinylated proteins can then be purified or selectively detected on a blot
using the very strong and specific interaction between biotin and streptavidin.

8.5.3 Identification of proteins subject to nNOS-dependent S-nitrosylation

This experiment illustrates the selectivity of protein-S-nitrosylation in brain tissue from mice due to
activation of neuronal NOS. The role of nNOS was ascertained by comparing the extent of
nitrosylation between wild-type and nNOS k.o. mice.
After extracting the proteins from homogenized tissue, S-nitrosyl groups were converted to biotin
derivatives as outlined above, and selectively detected on blots using labeled streptavidin. Total
protein (nitrosylated or not) was detected with antibodies and seems indistinguishable between
wild-type and nNOS k.o. mice in all cases. With each protein, the extent of S-nitrosylation is
represented by the intensity of the third sample relative to the first one.
In all cases, S-nitrosylation is caused by nNOS, as shown by its absence in the knockout mice. The
extent of nitrosylation is particularly strong with the NMDA receptor, and indeed this receptorone
of the ligand-gated glutamate receptors (see section 6.11)is known to be inhibited by Snitrosylation. Glyceraldehyde-3-dehydrogenase (GAPDH) is also quite strongly affected.
Intriguingly, this protein does not only serve in its catalytic role in glycolysis but moonlights as a
signaling molecule in the control of apoptosis (programmed cell death). Figure adapted from [1].

8.6 Role of NO and iNOS in the killing of microbes by phagocytes

Microscopic image from pathorama.ch

NO released by eNOS and nNOS serves signaling roles and is subject to rapid, calcium-dependent
regulation. In contrast, NO produced in cells of the immune system primarily serves as an
inflammatory effector. Its production doesnt need to occur as rapidly but must be sustained for
longer, and accordingly is regulated by transcriptional induction of the enzyme rather than calcium
and calmodulin. This form of the enzyme is therefore called inducible NOS (iNOS).
The antimicrobial effect of NO is strongest in combination with reactive oxygen species. In
phagocytosis, microbes are ingested by granulocytes or macrophages and wind up inside
intracellular vesicles called phagosomes. Peroxisomes then fuse to the phagosomes, releasing
superoxide (produced by NADPH oxidase) and H2O2 (produced by superoxide dismutase).
Concomitantly, iNOS is activated in the cytosol. NO enters the phagosomes, where it may combine
with superoxide to form peroxynitrite, which is strongly microbicidal. Alternatively, NO may
diffuse across the microbial cell wall to wreak havoc inside.
As with reactive oxygen species, the production of NO by inflammatory cells is a mixed blessing
important in antimicrobial defense, but destructive in rheumatic or other autoimmune disease. In
severe, systemic infection (septicemia), the massive release of NO by immune cells causes
relaxation of the vasculature, leading to a dangerous drop in blood pressure (septic shock).
Therefore, pharmacological inhibition of iNOS is of great medical interest.

8.7 NO-releasing drugs

Release of NO from nitroglycerin and ISDN by human cytochrome P450 isoforms
9

NO release by molsidomine

The drugs shown in this slide are used in the treatment of hypertension and of stenotic coronary
artery disease. The first drug to be so employed was nitroglycerin, which is more commonly known
as an explosive. Its therapeutic properties were discovered when workers exposed to nitroglycerin
fumes at Mr. Alfred Nobels factory complained about headaches recurring at the beginning of each
work week (headaches are often caused by dilating blood vessels in the skull).
Nitric oxide is released from nitroglycerin by cytochrome P450 enzymes. The drug is promptly
absorbed across mucous membranes, and sublingual application of spray or droplets is used by
patients with acute attacks of angina pectoris. Isosorbide dinitrate is used by the same group of
patients, but has a slower and longer lasting effect than nitroglycerin and is used orally for sustained
therapy.
Sodium nitroprusside releases NO spontaneously, without enzymatic catalysis. It is the most
powerful NO-releasing drug and is used, by intravenous infusion, in hypertensive crisis and other
emergencies involving dangerously high blood pressure. Somewhat counterintuitively, it is also
used in hemodynamic shock, which involves extremely low blood pressure. In this condition,
perfusion of the kidneys and other interior organs is reduced by autonomic reflexes to a dangerous
extent. After stabilizing blood pressure with other means, nitroprusside is used to break through this
reflex blockade and restore perfusion to the endangered organs.
In addition to NO, nitroprusside also releases cyanide. This is potentially toxic but can be
neutralized by the simultaneous application of sodium thiosulfate, which reacts with cyanide to
form thiocyanate.

8.7.1Release of NO from nitroglycerin and ISDN by human cytochrome P450 isoforms

10

While both nitroglycerin and isosorbide dinitrate are metabolized by cytochrome P450 enzymes, a
more detailed analysis shows that the enzyme isoforms with the strongest activity toward them are
somewhat different: CYP2E1 is highly active with nitroglycerin, whereas CYP3A4 is more active
with ISDN. Both drugs are converted efficiently by CYP2J2, which is strongly expressed in blood
vessels and is likely important in the prompt clinical effect of nitroglycerin.
As we have seen, cytochrome P450 enzymes contain heme. Like the heme in sGC, the one in
cytochrome P450 may bind NO; this will inactivate the enzyme. Inactivation of cytochrome P450
by NO released from drugs contributes to nitrate tolerance, which develops upon prolonged drug
application and can only be broken by pausing the use of these drugs.*
In the experiment shown, the CYP isoforms were recombinantly expressed in yeast. Experimental
systems such as this one are often used to study the susceptibility of novel drug molecules to
metabolism during preclinical development. Figure prepared from original data in [1].

8.7.2 NO release by molsidomine

11

Molsidomine is an alternative NO-releasing drug that does not require metabolism by cytochrome
P450, and therefore is less prone to the development of tolerance. The left panel shows the
mechanism of NO release: Enzymatic hydrolysis of molsidomine yields linsidomine, which then
decomposes spontaneously. The final step depends on O2 and yields both NO and superoxide.
As we had seen above (slide 8.6), superoxide can combine with NO to form peroxynitrite.
Accordingly, in order to render the NO released by linsidomine available for the activation of
guanylate cyclase, superoxide must be scavenged. This is illustrated in the experiment in the right
panel, which shows that appreciable amounts of cGMP are formed downstream of linsidomine
decay only in the presence of scavenging agents such as superoxide dismutase (SOD) or glutathione
(GSH). Figure prepared from original data in [2].
8.8 Antiinflammatory effect of iNOS inhibition

The activity of iNOS in inflammation is potentially destructive and contributes to tissue damage in
rheumatism and other autoimmune diseases; therefore, inhibiting iNOS is potentially useful. To
make this work, it is important to avoid inhibition of eNOS and nNOS. While most NOS inhibitors,
12

such as l-N-methyl-arginine-methylester (l-NAME) inhibit all NOS isoforms, some, like the
experimental drug GW274150, are selective for iNOS.
In the experiment on the right, the effectiveness of GW274150 was studied in a mouse model of
inflammation. Collagen-induced arthritis can be provoked in mice by injecting their paws with
bovine collagen type II, to which the mice then mount an immune reaction that leads to swelling
and inflammation.
After injection on day 0, the mice take three weeks to produce antibodies and develop
inflammation. The intensity of this inflammation can be measured by the degree of paw swelling.
Genetic knockout of iNOS and the experimental drug are equally effective in mitigating
inflammation. Figure prepared from original data in [1].
8.8.1 Structure and mode of action of sildenafil (Viagra)

While NO-releasing drugs influence the contractile activity of smooth muscle cells by increasing
cGMP, an equally viable approach should be the inhibition of cGMP degradation by
phosphodiesterase. This rationale led to the development of sildenafil, which was originally
intended as yet another cardiovascular drug for the elderly.
As it turned out, the drug proved very popular with the old folks alright, but not entirely for the
intended reasons. Nevertheless, in addition to its well-known effect on male potency, the drug also
does promote vasorelaxation, which can prove dangerous when used in combination NO-releasing
drugs or similar. In the pragmatic but mostly involuntarily humorous nation of Switzerland, this has
prompted the training of prostitutes in the use of defibrillators.

13