SYMPOSIUM
"MOLECULAR ADVANCES IN NITRIC OXIDE RESEARCH"
University of Maryland Baltimore County
Department of Chemistry and Biochemistry
|
DATE:
November 17, 2010
TIME: 8:30am-3:45pm
LOCATION: UMBC, Chemistry Building
Conference
Room 120
1000
Hilltop Circle
Baltimore,
MD 21250
The symposium is free and open to the public. Refreshments will be provided. Lunch is not.
For lunch possibilities on campus (cash or credit card), please visit Where to eat
RSVP is required before October 30th for
parking on campus
Please email Elsa Garcin or Jim Fishbein
at NOSymposium@gmail.com to RSVP
PROGRAM
8:30 AM Opening
remarks by Geoffrey P. Summers
8:40 AM Keynote
lecture by Prof. Salvador Moncada
"Nitric oxide and mitochondrial interactions: physiology and pathophysiology"
9:40 AM
David Wink
"Inducible Nitric Oxide Synthase and Cycloxygenase-2 in Cancer. Identification of Potential Molecular Pathways and
Targets that Lead to Poor Prognosis."
10:20 AM Coffee
Break
10:40 AM
Larry Keefer
"Nitric oxide-based drug development"
11:20 AM Nazareno Paolocci
"Nitroxyl (HNO) ...
From Heaven to Heart: Stardust or a New Tool against
Heart Failure?"
12:00pm Lunch
Break
1:30 PM
David Roberts
"Thrombospondin-1
signaling via CD47 in injury and stress responses"
2:10PM
Jeffrey Isenberg
"Thrombospondin-1 supports blood pressure by limiting
eNOS activation and endothelial-dependent
vasorelaxation"
2:50pm
Gerald Rameau
"Nitric
Oxide Regulation of Glucose Transporter 3 (GLUT3)"
3:10pm
Elsa Garcin
"Structural
studies of a mammalian soluble
guanylate cyclase.
Lessons learnt from nitric oxide synthase"
3:30pm Travel
Awards
3:40pm Closing
remarks
3:45pm
Refreshments
and closing of the symposium
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SPEAKER ABSTRACTS
[Click on
the speaker name to view biography]
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KEYNOTE
PRESENTATION
Nitric oxide and mitochondrial interactions: physiology and pathophysiology
Director
of the Wolfson Institute for Biomedical Research,
University College London, London UK
At physiological concentrations nitric oxide (NO)
inhibits mitochondrial cytochrome c oxidase in competition with oxygen. We have developed a
technique based on visible light spectroscopy and used it to demonstrate that endogenous
NO enhances reduction of the electron transport chain, thus enabling cells to
maintain their VO2 at low oxygen concentrations. This favors the
release of superoxide anion, which initiates the transcriptional activation of
NF-kB as an early stress signaling response. We have also used this
technique to demonstrate that NO is inactivated by cytochrome
c oxidase in its oxidised
state and have proposed that cessation of such inactivation at low oxygen
concentrations may account for hypoxic vasodilatation. Many cells respond to a decrease in oxygen
availability via stabilisation of hypoxia-inducible
factor-1a (HIF-1a), whose accumulation is normally prevented by the
action of prolyl hydroxylases.
We have found that inhibition of mitochondrial respiration by low
concentrations of NO leads to inhibition of HIF-1a stabilisation.
This prevents the cell from registering hypoxia at low oxygen concentrations,
which would otherwise result in upregulation of defensive
genes, including those for glycolysis and
angiogenesis. Furthermore, inhibition of mitochondrial respiration in hypoxia
leads to redistribution of available oxygen toward non-respiratory
oxygen-dependent targets. In addition to its
interaction with cytochrome c oxidase, NO can signal
for mitochondrial biogenesis via a cyclic GMP-dependent mechanism. Furthermore, Increases in
NO beyond physiological levels lead to persistent inhibition of other key
enzymes in the mitochondria and this may account for NO-dependent initiation of
cell pathology.
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Inducible Nitric Oxide Synthase and Cycloxygenase-2 in Cancer. Identification of Potential Molecular Pathways and
Targets that Lead to Poor Prognosis
National
Institutes of Health, National Cancer Institute, Radiation Biology Branch,
Bethesda USA
Epidemiological studies have found that inflammatory
proteins iNOS and COX-2 are poor prognostic
indicators for many cancers. We
have been investigating the chemical mechanism of chemistry of NO and other
reactive species associated with biological mechanisms in cancer. These studies have shown that specific
concentrations of NO determine the pro or anti-tumorigenic
behavior. When prolonged exposure to µM amounts of NO, there is an increase in
the phosphorylation of p53 and cystostasis. However, when cells are exposed to 100 nM of NO, there is increase in protumorigenic
molecular pathways such as MAPK, pAkt and HIF1a.
Several of these pathways are also activated by PGE2. In ER negative breast
cancer patients we found that if either iNOS
and COX-2 was highly expressed there was a decrease in survival. When both were present there was a
dramatic decreased survival. From
this epidemiological data we have been able to develop cellular models to
determine if we can find compounds that will reverse these mechanisms that
result in poor phenotypes. We have
found a class of thiol and HNO-based compounds that
activate a tumor suppressor protein reversing the molecular pathways associated
with iNOS and COX-2 in ER negative breast
cancer. This talk will focus on
the understanding the chemical biology of nitric oxide and how it increases
cancer risk and describe some potential new molecular targets for the treatment
of cancer. -------------------------------------------------------------------------------------------------- Nitric oxide-based drug development National
Cancer Institute Frederick, Laboratory of Comparative Carcinogenesis, Frederick
USA Nitric oxide (NO) is a
central player in the inflammatory process. To investigate its many roles in
normal as well as pathophysiology, caged NO donors of the diazeniumdiolate class
(also known as NONOates) have come into wide use as research tools, offering a
range of reliable half-lives from 2 seconds to 20 hours for spontaneously
generating authentic NO into aqueous media at physiological temperature and pH.
Current work is aimed at exploring the utility of this caged NO chemistry in
designing improved drugs and biomedical devices. My presentation will highlight
a selection of recent advances in this area. -------------------------------------------------------------------------------------------------- Nitroxyl (HNO) ...
From Heaven to Heart: Stardust or a New Tool against
Heart Failure? Dr. Nazareno Paolocci Johns
Hopkins Medicine, Johns Hopkins Heart and Vascular Institute, Baltimore USA Nitroxyl (HNO) is the one
electron-reduction product of NO.. HNO was
first discovered in the clouds of interstellar space. Yet its endogenous formation
in mammals still awaits definitive proof. However, the pharmacological
properties of HNO donors are emerging as neatly distinct from those exhibited
by NO., nitrogen-related or oxygen-derived reactive species,
particularly in the cardiovascular system. In vivo, HNO elicits both
vasodilation and cardiac function enhancement, namely positive inotropy/lusitropy.
In vitro, HNO cardiac properties are fully independent from cGMP/PKG and
cAMP/PKA signaling, but relying on the availability of critical thiols, namely
cysteines, situated in key components of the electro-contraction coupling
machinery of the heart. HNO enhances Ca2+ cycling at the
sarcoplasmic reticulum (SR) level without affecting Ca2+ level at
rest. Moreover, it sensitizes myofilaments to Ca2+. The involvement
of critical "redox-switches" in HNO cardiac effects is supported by the fact that
HNO action is prevented when the intracellular amount of free-floating thiols rises
(or reversed when more reducing equivalents are provided) and by
cysteine-to-alanine mutagenesis experiments. The fact that HNO action is fully
retained in vivo hearts and in vitro myocytes suffering from heart
failure (HF), both typically displaying increased oxidative stress, suggests that
HNO may target a selected population of highly reactive cysteines, not easily
accessible for other ROS/RNS. In conclusion, HNO donors may be advantageous for
treating HF patients that have impaired cardiac contraction, volume overload and
high peripheral vascular resistance, and in whose hearts oxidative stress and
altered Ca2+ handling coexist. -------------------------------------------------------------------------------------------------- Thrombospondin-1
signaling via CD47 in injury and stress responses National
Institutes of Health, National Cancer Institute, Laboratory of Pathology,
Bethesda USA The thrombospondin-1
receptor CD47 controls angiogenesis, vascular tone, and survival of ischemic
stress by regulating nitric oxide (NO) signaling. CD47 ligation redundantly inhibits signaling upstream and
downstream of NO. Soft tissues in thrombospondin-1- and CD47-null mice are
highly resistant to ischemia and ischemia/reperfusion injuries. Remarkably, this protection extends to
high-dose radiation injury. Similar
protection can be achieved in wild type animals by treatment with antagonists
of thrombospondin-1-CD47 interaction.
Tumors in mice treated with CD47-blocking agents, however, become more
sensitive to radiation. Some of these therapeutic activities can be reproduced by NO donors
or agents that elevate tissue cGMP levels, but
this is not the case for ischemia/reperfusion and radiation injury
responses. This implies that
targets of CD47 other than the NO/cGMP pathway are
involved. We are examining the
role of additional signaling pathways in vascular and immune cells that are
regulated by CD47 in order to optimize these therapeutic activities. -------------------------------------------------------------------------------------------------- Thrombospondin-1 supports blood pressure by limiting eNOS activation and endothelial-dependent vasorelaxation University
of Pittsburgh, Vascular Medicine Institute, Pittsburgh USA Thrombospondin-1 (TSP1), via its necessary receptor
CD47, inhibits nitric oxide (NO)-stimulated soluble guanylate
cyclase activation in vascular smooth muscle cells,
and TSP1-null mice have increased shear-dependent blood flow compared with
wild-type mice. Yet, the endothelial basement membrane should in theory
function as a barrier to diffusion of soluble TSP1 into the arterial smooth
muscle cell layer. These findings suggested that endothelial-dependent
differences in blood flow in TSP1-null mice may be the
result of direct modulation of endothelial NO synthase
(eNOS) activation by circulating TSP1. Here we tested
the hypothesis that TSP1 inhibits eNOS activation and
endothelial-dependent arterial relaxation. Acetylcholine (ACh)-stimulated
activation of eNOS and agonist-driven calcium
transients in endothelial cells were inhibited by TSP1. TSP1 also inhibited eNOS phosphorylation at
serine1177. TSP1 treatment of the endothelium of wild-type
and TSP1-null but not CD47-null arteries inhibited ACh-stimulated
relaxation. TSP1-null vessels demonstrated greater endothelial-dependent vasorelaxation compared with the wild type. Conversely,
TSP1-null arteries demonstrated less vasoconstriction to phenylephrine
compared with the wild type, which was corrected upon inhibition of eNOS. In TSP1-null mice, intravenous TSP1 blocked ACh-stimulated decreases in blood pressure, and both
intravenous TSP1 and a CD47 agonist antibody acutely elevated blood pressure in
mice.TSP1, via CD47, inhibits eNOS
activation and endothelial-dependent arterial relaxation and limits ACh-driven decreases in blood pressure. Conversely,
intravenous TSP1 and a CD47 antibody increase blood pressure. These findings
suggest that circulating TSP1, by limiting endogenous NO production, functions
as a pressor agent supporting blood pressure. -------------------------------------------------------------------------------------------------- Nitric Oxide Regulation of Glucose Transporter 3 (GLUT3)
Morgan State University, Baltimore USA The
glucose transporter type 3 (GLUT3) regulates glucose uptake, which provides neurons
with an energy substrate. However, the mechanism by which synaptic activity regulates
GLUT3 trafficking has not been elucidated. To examine activity dependent
regulation of GLUT3, primary cultured neurons were stimulated with bicuculline, which induced synaptic activation of N-Methyl-D-Aspartate receptor (NMDAR). Stimulation of NMDAR increases
the production of NO by neuronal nitric oxide synthase
(nNOS). This regulation is achieved by Ca2+-Calmodulin
binding and phosphorylation. nNOS is phosphorylated by Akt at S1412 (positive regulation) and by CaMKII at S847 (negative regulation). We found that activation
of NMDAR induces a program of positive and negative phosphorylation
of nNOS, which suggests physiological function. This
activation increased surface GLUT3 via a NO/cGK
pathway that correlated with increased glucose uptake. In total, our results
suggest a mechanism that regulates surface GLUT3 that may serve homeostatic
strategies allowing neuronal cells to meet the changing energy demands brought
about by increased synaptic activity. -------------------------------------------------------------------------------------------------- Structural studies of a mammalian soluble guanylate cyclase. Lessons learnt from nitric oxide synthase University
of Maryland Baltimore County, Department of Chemistry and Biochemistry,
Baltimore USA Soluble
guanylate cyclase (sGC) is the direct sensor and mediator of nitric oxide (NO) signal
transduction via cyclic GMP (cGMP). NO-induced vasodilation depends primarily on the activation of sGC, which produces cGMP whose
effects are mediated by cGMP-dependent kinases, ion channels, and phosphodiesterases.
Compounds
that activate cGMP production by sGC
have outstanding clinical potential for treating cardiovascular and other
diseases due to impaired blood flow. To elucidate the structural details of sGC assembly and determine the dynamic events associated
with NO-induced sGC activation, we have initiated a
multidisciplinary approach using biochemical, mutagenesis studies, combined with
structural methods. Working with mammalian sGC
is not trivial. First, obtaining large quantities of pure protein has been a
major bottleneck in the sGC field. Second, purification is
complicated by (i) the fact that the sGC heme is extremely labile,
(ii) the presence of 34 cysteine residues, and (iii)
sensitivity to proteolysis. Nitric oxide
synthase posed similar challenges to studying its
assembly, catalysis and regulation, therefore our
laboratory will apply lessons learned from the biophysical characterization of
NOS to shed light on sGC structure and function. Our first step was to design a bacterial overexpression system for bovine sGC.
To our knowledge, we have developed the first bacterial overexpression system yielding soluble and active
full-length mammalian sGC, as well as truncated
constructs. Here, I will present our recent advances towards structural studies
of bovine sGC. -------------------------------------------------------------------------------------------------- Sponsor: DOW Chemical Company
Organizers: Elsa Garcin & Jim Fishbein