Transcript Physiology of the metabolic gases Piantadosi 2014

PHYSIOLOGY OF THE
METABOLIC GASES
Claude A. Piantadosi, M.D.
Professor of Medicine
Director, Center for Hyperbaric Medicine
And Environmental Physiology
THE METABOLIC GASES
•
Questions for today
• What are the physiological gases?
• What is physiological O2 sensing?
• What is hypoxic vasodilation?
• What is ROS signaling?
• When does ROS production become pathological?
THE METABOLIC GASES
•
Metabolic gases
• CO2
• O2
• NO
• CO
• H 2S
• ROS
•
Inert gases
• N2
• Ar
• He
• H2
THE METABOLIC GASES
•
Metabolic Gas
•
•
•
•
•
CO2
O2
NO
CO
H 2S
Physiology
Toxicity
pH/Vasodilation
Respiration/
Vasoconstriction
Vasodilation
Vasodilation
Vasodilation
Narcosis
Oxidation
Nitration
Asphyxia
Asphyxia
Metabolic gas concentrations vary not just with solubility and partial
pressure, but with quantity and number of binding targets in cells and
tissues— this defines their reactivity
THE METABOLIC GASES
Physiological, adaptive, and toxic effects of all metabolic
gases depend on dose and time
Concentration
•
Toxic
Adaptive
Physiological
Time
CARRIAGE OF CO2 IN BLOOD

CO2 is produced by mitochondrial TCA cycle and transported in
the blood from tissue to lungs in three ways:




Dissolved in solution
Buffered with water as carbonic acid
Bound to proteins, particularly hemoglobin
About 75% of CO2 is transported in RBCs and 25% in plasma
CARRIAGE OF CO2 IN BLOOD*
100%
100%
5%
Carbamino
90%
HCO3-
60%
Dissolved
10%
30%
5%
0%
Arterial
Venous
0%
CO2 distribution in
arterial & venous blood

*Approximate values (CO2 content of blood is influenced by
hemoglobin concentration and saturation, 2, 3-DPG, and pH).
Estimates include bicarbonate and CO2 inside the RBC
THE METABOLIC GASES
•
CO2 transport: RBC plays a critical role
Tissue
RBC
CO2 production by tissues
favors O2 unloading
CO2
SaO2
+ H2O
H2CO3
CA
Bohr effect
H+ +
PaO2
CA
HCO3
CO2 +H2O
CO2——CO2
Band 3
Plasma
Cl-
HCO3Lungs
THE METABOLIC GASES: CO2
Deoxyhemoglobin
60
•
CO2 dissociation
curve of blood
O2 dissociation
curve of blood
Bohr Effect
O2 or CO2 content
(mL/100 mL)
•
Haldane effect
Oxyhemoglobin
40
HbO2
20
Dissolved CO2
Dissolved O2
0
0
50
100
150
PO2 or PCO2 (mmHg)
OXYGEN TRANSPORT TO TISSUES
•
The O2 cascade
150
PO2
(mmHg)
100
50
0
Air
Alveolus
Artery
Capillary Mitochondrion
THE METABOLIC GASES
•
Mitochondrial sink for cellular O2 diffusion
 Increasing oxygen affinity
100
100
NADH
or
Cyt a,a3
(% Oxidation)
NADH
Cyt a,a3
50
Mb
State 4
0
10-8
50
Hb
State 3
10-7
0
10-6
10-5
10-4
Oxygen concentration (M)
10-3
Hb or Mb
(% Oxygenation)
THE MAIN CONSUMER OF O2
Mitochondria ~95%
Respiration:
6O2+ C6H12O6+ 30Pi2-+ 30ADP3-+ 30H+  6CO2+ 30ATP4-+ 36H2O
THE METABOLIC GASES
•
Rate of O2 consumption depends on the MichaelisMenton constant:
Vmax
VO2
50%
Diving and hyperbaric range
3-4 ATA
KM
•
[O2]
O2 is rarely, if ever, rate-limiting under hyperbaric
conditions
PHYSICAL PROCESSES OF O2 TRANSPORT
•
•
•
•
•
•
Diffusion— alveolus to blood
Chemical combination— hemoglobin
Convective transport— tissues
Chemical release— hemoglobin
Diffusion— blood plasma to cells
Chemical reduction to water— mitochondria
THE METABOLIC GASES
•
HBO2 and the HbO2 dissociation curve
25
Dissolved
oxygen
20
Normal AVO2 difference
CaO2 15
(ml/dl)
10
5
0
0 100
500
1000
PO2 (mmHg)
1500
THE METABOLIC GASES
•
Arterial O2 content— Sea level Air O2 and HBO2
at 2.5 ATA
CaO2 = 1.34 ml/g [Hb](SaO2) + 0.003 ml O2/dl/mmHg
= 1.34ml/g [15.0g/dl](1.0)
+ 0.003 ml/dl/mmHg O2x100 mmHg
= 1.34ml/g [15.0g/dl](1.0) + 0.3 ml O2
= 20 ml O2/dl + 0. 3 ml O2/dl= 20.3 ml O2/dl (Air)
= 20 ml O2/dl + 2. 1 ml O2/dl= 22.1 ml O2/dl (O2)
= 20 ml O2/dl + 5.4 ml O2/dl= 25.4 ml O2/dl
Dissolved Oxygen
THE METABOLIC GASES
•
Determinants of PO2 in tissue
•
•
•
•
•
•
Capillary hematocrit
Position of hemoglobin O2 dissociation curve
Adequacy and uniformity of perfusion
O2 shunting
Capillary transit time
Rate of cell respiration
THE METABOLIC GASES
HBO2
THE METABOLIC GASES
•
O2 diffusion into tissue—Krogh cylinder model
Venous
r
Arterial
r
A
PO2
Dead corner
V
PO2
VO2 max
V
r
}
}
r
A
THE METABOLIC GASES
•
O2 diffusion into tissues
Venous
Air r = 12mm
HBO2 r = 60mm
r
r
PaO2
Air
r
HBO2
Arterial
Air r = 60mm
HBO2 r = 300mm
OTHER O2 CONSUMERS
e-
e-
e-
e-
ROS generation: O2  .O2-  H2O2  .OH  2H2O
+2H+
+2H+
NADPH
Nitric oxide synthase: O2 + L-arginine  NO.. + L-citrulline
NADPH
Heme oxygenases: O2+ heme  CO+ Fe + biliverdin
Cytochrome P450: O2 + RH + 2H+ +
NADPH
2e–  ROH
+ H2O
NADPH oxidases:: 2O2 + NADPH NADP+ + 2.O2- + H+
~5%
THE METABOLIC GASES
•
High PO2 promotes ROS generation
Protein oxidation
• Thiol (SH) oxidation
• Lipid peroxidation
• DNA oxidation
•
e-
e-
e-
e-
O2  .O2-  H2O2  .OH  H2O
+2H+
REDOX SIGNALING BY ROS
Physiological
States
Pathological
States
Low
ROS
Levels
High
ROS
Levels
Localized
De-localized
Cell proliferation
Adaptation to stress
Promote injury repair
Change cell phenotype
Kills pathogens
Interferes with cell function
Blocks cell repair
Causes apoptosis/necrosis
Promotes tissue injury
Chronic anti-oxidant therapy
ineffective or harmful
Chronic anti-oxidant therapy
more likely to be effective
THE METABOLIC GASES:
VASCULAR CONTROL BY NO
•
Many vascular control events require NO production
•
Examples:
•
•
CO2-induced vasodilation
• NO plays a permissive role
O2-induced vasoconstriction
• Profound vasoconstriction at PO2 >500 mmHg
• Arterial and venous vessels
• Reduces cerebral, retinal, and renal blood flow
• Limits inert gas clearance from tissues
THE METABOLIC GASES
•
O2-induced vasoconstriction
• HBO2 decreases vasodilator activity of NO by generating
superoxide (.O2-)
• .O2- inactivates NO forming the strong oxidant peroxynitrite
(ONOO-)
• Hyperoxia prevents allosteric unloading of NO from RBCs by
SNO-hemoglobin
METABOLIC GASES:
•
NO
NOS isoforms
•
•
•
•
nNOS (type I constitutive)
iNOS (type II inducible)
eNOS (type III constitutive and inducible)
mtNOS (nNOS variant)
Vascular NOS:
eNOS
iNOS
nNOS
METABOLIC GASES:
•
NO
O2-induced vasoconstriction
O2
e-
Reactive nitrogen species (RNS)
NOS
NO. + .O2 -
ONOO-
(superoxide)
(peroxynitrite)
H+
ONOOH
(peroxynitrous acid)
(6.7 X 109 M-1 s-1)
NO2 + OH.
Dilation
Constriction
Toxicity
METABOLIC GASES:
•
NO
The L-arginine-nitric oxide pathway
L-arginine
L-ornithine
Arginase
L-arginine + O2
Arginosuccinate
NOS
GTP
(-)
cGMP
NO-heme-sGC
NG-OH-L-arginine
L-citrulline + NO
NO
R-SH
R-SNO + H+
Effector cell
Target cell
(endothelial)
(smooth muscle)
METABOLIC GASES:
•
NO
Multiple levels of eNOS regulation
•
•
•
•
•
Transcriptional control
Translational control
• Cytokine-driven mRNA degradation
Post-translational modification
• Phosphorylation/ Myristoylation/ Palmitoylation
Protein-protein interactions (enzyme localization)
• Calmodulin/ Hsp90/ Caveolin
Uncoupling
• BH4/ L-arginine deficiency
Fleming I. Molecular mechanisms underlying the activation of eNOS.
Pflugers Arch. May;459(6):793-806, 2010
H2O2 IS A PLEIOTROPIC VASODILATOR
•
H2O2 mediates endotheliumdependent or independent
vasorelaxation
•
•
•
•
•
•
NO-dependent
NO- independent
H2O2 activates eNOS in large vessels,
leading to eNOS-dependent relaxation
In small vessels, e.g. coronary
arterioles, mitochondrial-derived H2O2
is responsible for flow-mediated
vasodilation (NO-independent)
In disease, e.g. atherosclerosis and
hypertension, H2O2 produced by large
vessels mediates compensatory,
endothelial-dependent, but NO.independent relaxation
H2O2 may cause endotheliumindependent relaxation via catalase
compound I activation of smooth
muscle cGMP
METABOLIC GASES:
•
CO
Carboxyhemoglobin (COHb) derived from endogenous and exogenous sources
Condition
COHb
Normal
1-2%
Pregnancy
2-4%
Hemolytic anemia
2-6%
Cigarette smoking
4-5% /pack/day
CO poisoning
20-50%
METABOLIC GASES:
•
CO
CO decreases blood O2 content and tissue PO2
20
100% HbO2
AVDO2
15
CaO2 or CvO2
(ml/dl)
10
50% COHb
AVDO2
5
0
0
25
50
PaO2 (mm Hg)
75
100
METABOLIC GASES
•
CO and CO body stores
OSHA 8-hour exposure limit is 50 ppm
• Endogenous CO production by HO reflects ~ 1-5 ppm
•
Intravascular
Extravascular
Myoglobin
CO
Alveolar gas
COHb
Hemoprotein
enzymes
Endogenous
CO production
Metabolism
to CO2
Hypoxia
METABOLIC GASES:

CO
Dual mechanism of CO poisoning

Chemical asphyxia (CO hypoxia)
 COHb
has increased O2 affinity
Tissue hypoxia
 COHb does not carry O2
 Haldane’s First Law:
[COHb]/[HbO2]= M (PCO/PO2), M=220

Cellular poisoning—heme protein binding
 Warburg constant: K= (n/1-n)(CO/ O2)
Where n, the fraction bound to CO, is equal to 0.5
K is the ratio of CO:O2 to half-saturate the binding site
REDOX-REGULATION OF
MITOCHONDRIAL BIOGENESIS
Sepsis-induced inflammation
-SH
oxidation
Ub
TLRs
Nrf2 Proteasome
Nrf2
Keap1
HO-1/CO
PI3K/ Akt1
PTEN
GSK3b
NFkB
Nrf2
P
P
P
MyD88
Nrf2
ARE
Nrf2
ARE
Hmox1
NO
NRF-1
NRF-1
PGC-1a
Mitochondrial biogenesis
Anti-oxidant enzyme induction
Anti-apoptosis (Bcl2)
Counter-inflammation (IL-10)
Mitophagy (p62)
P
NRF-1
Nucleus
Heme oxygenase-1 regulates cardiac mitochondrial biogenesis via Nrf2-mediated
transcriptional control of nuclear respiratory factor-1.
Piantadosi CA, Carraway MS, Babiker A, Suliman HB.
Circ Res. 2008 Nov 21;103(11):1232-40.
THE METABOLIC GASES
•
CO binds iron and other transition metals
allowing it to interact with ROS and NO
NO
Fe III
ONOO-
e-
O2
.O 2
Pro-oxidant
CO
RSH
.O 2
Fe II
H2 O2
.OH
Anti-oxidant
METABOLIC GASES:
•
H2 S
Hydrogen sulfide
•
•
•
•
Sewer gas (rotten eggs)
Poisons mitochondrial ETC at high levels
Generated enzymatically by cells and plays several
physiological roles
Relationship to O2 mainly involve sulfide oxidation
HYDROGEN SULFIDE CHEMOSYNTHESIS
•
Chemosynthesis
•
Biological conversion of one or more carbons (usually CO2 or
CH4) into organic matter by oxidation of inorganic molecules
(H2 or H2S) or CH4 as a source of energy, rather than by
sunlight (photosynthesis)
Hydrogen sulfide chemosynthesis:
6CO2 + 6H2O + 3H2S = C6H12O6 + 3H2SO4
•
Some bacteria do this, e.g. purple sulfur bacteria, instead of
photosynthetic release of O2
•
•
Yellow sulfur globules produced that are visible in the cell
Proposed that chemosynthesis may support life below the
surfaces of Mars, and Jupiter's moon Europa
METABOLIC GASES:
H2 S
Kabil O, Motl N, Banerjee R. H2S and its role in
redox signaling. Biochim Biophys Acta 2014 Jan 11
METABOLIC GASES:
•
H2 S
Enzymatic H2S Production
• 3-mercaptopyruvate sulfurtransferase (MST)
• Cystathione gamma lyase (CSE)
• Cystathionine beta-synthase (CBS)
• CBS normally condenses serine and homocysteine to
cystathionine:
L-serine + L-homocysteine = L-cystathionine + H2O
•Only pyridoxal phosphate-dependent enzyme that contains a heme co-factor that
functions as a redox sensor; modulates activity in response to redox potential.
•Resting form of CBS has ferrous heme (Fe II) that is activated under oxidizing
conditions by conversion to ferric state
•Fe (II) form is inhibited by CO or NO binding; activity doubles when Fe (II) Fe (III)
METABOLIC GASES:

H2 S
Controversies surround the sometimes conflicting
effects of H2S (e.g. both pro- and antiinflammatory)

Highlights problems associated with interpreting
studies
Very wide concentration range of H2S
 Technical challenges of handling a redox-active gas


Multiple mechanisms of H2S-based signaling
Protein persulfidation
 Sulfhydration of electrophiles
 Interaction with S-nitrosothiols
 Interaction with metal centers

Sulfide biosynthesis
H2S SYNTHESIS
AND DEGRADATION



Tissue H2S concentration is
low 10–30 nM except in aorta
Sulfur flux into H2S in
murine liver is comparable to
GSH (6–10 mM at steadystate)
Thus, sulfide clearance rate
must be high to account for
low steady-state H2S
concentrations
Sulfide clearance
Kabil O, Motl N, Banerjee R. H2S and its role in
redox signaling. Biochim Biophys Acta 2014 Jan 11
METABOLIC GASES:
H2 S
O2
Storage
Iron sulfide (Fe-S)
Sulfane sulfur
Polysulfides
O2
Cytochrome oxidase
O2
Sulfmyoglobin
Biosynthesis
CBS
CSE
MST
Interactions
Neuromodulation
Muscle relaxation
Hibernation-like state
Cysteine H2S
O2
Degradation
Oxidation
Methylation
Sulfhemoglobin
NMDA receptor
KATP channel
H2S is degraded mainly in mitochondria through a series
of oxidations that convert the gas to sulfite (SO3-2),
thiosulfate (S2O3-2), and sulfate (SO4-2 )
Olson KR, Whitfield NL (2010) Hydrogen sulfide and oxygen sensing in the cardiovascular system.
Antioxid Redox Signal 12:1219–1234.
THE METABOLIC GASES

Summary

O2’s role is not limited to aerobic metabolism, but is
involved in the production of and interactions with
other metabolic gases
Of the O2 used in the body, ~95% is reduced to H2O by
respiration
 Non-respiratory processes use ~5% (ROS, NO, and CO)
 An increase in tissue PO2 above that needed to support
respiration does not increase VO2, but does increase O2
utilization by the other processes (depending on Km)
 This may interfere with O2 regulation of these processes
 Excessive ROS production leads to delocalization of redox
signaling, and macromolecular damage (oxidative stress),
disordered repair and cell death
