Title: ParacrineAutocrine signaling molecules
1Paracrine/Autocrine signaling molecules local
hormones todays examples are Prostaglandins
Nitric Oxide Wed, 28th March 2003 Alan Brash,
Dept of Pharmacology, RRB 510
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3Furchgotts results
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5Nitric Oxide Synthase (NOS) are fusion proteins
with oxygenase and reductase domains The three
forms are
reductase domain
oxygenase domain
reductase domain
from Cayman Chemical catalogue, 2002
6- Two main "targets" of NO
- 1. Guanylate cyclase - NO activates G-cyclase
increases cGMP levels - 2. Reaction with superoxide - gives
peroxynitrite, a reactive toxic molecule
7Formation of Peroxynitrite
Superoxide is formed by "one electron reduction"
of O2 Two reducing equivalents converts O2 to
H2O2 (hydrogen peroxide) It takes four electrons
to reduce O2 completely to water (2 x
H2O) Superoxide is formed in high levels by an
enzyme, NADPH oxidase, in activated leukocytes,
and in small quantities in normal cells. In
leukocytes, iNOS gives high NO production
Peroxynitrite is one of the agents used by
leukocytes to kill microbes Oxidative damage to
normal tissue is marked by the peroxynitrite-induc
ed nitration of tyrosine in proteins
8Activation of Guanylate Cyclase by NO
9Smooth Muscle relaxation by NO and its
potentiation by Viagra
- NO activates G-Cyclase
- cGMP activates PKG and phosphorylations cause
s/m relaxation - PDE-5 eliminates cGMP
- Sildenafil blocks PDE-5 and hence potentiates
the vasodilator effect
from Corbin Francis, JBC 274, 13729 (1999)
10Cartoon of PDE-5 dimer Zinc is involved in cGMP
hydrolysis in the catalytic domain cGMP
activates enzymatic activity by binding in
the allosteric domain
from Corbin Francis, JBC 274, 13729 (1999)
11PDE-5 inhibition by Sildenafil (Viagra)
from Corbin Francis, JBC (1999)
12Nitric Oxide, Red Cells and Hemoglobin
- From the time of the original investigations on
the structure of EDRF - in the 1980-90s, it was known that interaction
with hemoglobin (Hb) - tended to shorten the half-life of NO (EDRF).
- Now there are two theories that each bring a
different physiological - relevance to this interaction the NO packaging
transport hypothesis - and the hemoglobin compartmentalization
hypothesis - The packaging hypothesis suggests that the Hb in
red cells helps - transport NO from lungs to tissues. A complex of
NO with thiols - (glutathione, GSH) helps in the transport in and
out of red cells. - An unrelated hypothesis suggests that
compartmentalization of Hb - in red cells is vital to protecting
endothelial-derived NO from destruction, - and that the release of Hb that occurs in sickle
cell disease has detrimental - effects because it destroys the vasodilator NO in
the circulation.
13Nitric Oxide, Red Cells and Hemoglobin the
packaging theory of Stamler et al
Overall effect is to increase tissue vasodilation
by release of NO see refs and reviews by Stamler
JS
Jia, Bonaventura Stamler (1996) Nature 380,
221-6 Gow Stamler (1998) Nature 391,
169-73 Stamler et al (1997) Science 276, 2034-7
14Nitric Oxide, Red Cells and Hemoglobin the
compartmentalization hypothesis
From Commentary by JC Liao, Nature Med 8, 1350
(Dec 2002) on main paper by Reiter CD Gladwin
MT Nature Med 8, 1383 (2002) The authors show
that free Hb in plasma, normally very low, is
high in sickle cell disease. They propose this
free Hb destroys NO and thus contributes to
ischemic episodes associated with sickle cell
disease.
Figure 1 NO bioactivity in normal blood and
sickle-cell blood. a, Cell-free zone in normal
blood preserves NO bioactivity. b, Hemoglobin
compartmentalized by normal RBCs. Membrane-associa
ted (boundary layer, cytoskeleton) diffusional
resistance effectively separates Hemoglobin from
NO. c, In sickle-cell blood, plasma hemoglobin
released by excessive intravascular hemolysis
consumes NO and destroys its bioactivity.
15SUMMING UP Nitric Oxide is a huge area of
research The term nitric oxide gets over
50,000 hits in PubMed, and nitric oxide and
review gets nearly 7000. Drugs that release NO
have been used for decades prior to
the identification of NO as a natural local
hormone and signaling molecule. As our
understanding of the biochemistry and physiology
improves, new targets for therapy will emerge,
most probably directed towards enhancing the
effects of NO in a more tissue selective manner.
16endocrine paracrine autocrine