The truth about the movement of NO across cell membranes
Jeffrey Garvin
Hypertension and Vascular Research DivisionDepartment of Internal Medicine
Henry Ford Hospital
Acetylcholine-induced EDRF release
NE
Ach -8 -7 -6 w
2 g
5 min
intact
rubbed
Ach -8 -7
-6 wNE
Furchgott et al., Nature 1980
NO synthesis
COOHHCNH2
CH2
CH2
CH2
NH CNH NH2
COOHHCNH2
CH2
CH2
CH2
NH CNOH
NH2
COOHHCNH2
CH2
CH2
CH2
NHCO
NH2
NO+
L-arginine L-citrulline
Why do we care about NO?
NO is involved in:
1. CNS function and cognition2. Cardiac contractility
3. Peripheral vascular resistance4. Respiration
5. Gut motility and ion absorption6. Renal perfusion and transport
7. Reproduction
Properties of NO
1. It is small.
2. It is non-polar.
3. It is RELATIVELY lipophilic with a partition coefficient of about 5.
4. It is a gas.
5. Its reactive (different from O2 and CO2).
soluble guanylatecyclase
NO
How many think NO diffuses through two bilayers
NOS 3
endothelial cell vascular smooth muscle cell
distance0
5
10
Energy profile of NO with distance based on partition coefficient
mem
bran
e
mem
bran
e
Arb
itra
ry e
ner
gy
un
its
guan
ylyl
cyc
lase
NONO
NO
NO
NO
NO
NONO
NO
NONO
NO
soluble guanylatecyclase
NOS 3NONO
NO
NO
NO
NO
NONO
NO
NONO
NO
NO
A slightly more “realistic” model of NO diffusion through bilayers
endothelial cell vascular smooth muscle cell
There have been no direct measurements of the NO permeability of any cell membrane!!!
There has been one calculation which is widely cited. This value of 76 cm/s was calculated based onsteady-state measurements of NO within an artificial membrane using 2 mM NO.
Free diffusion creates several problems:1. Free diffusion is relatively slow;2. The amount of NO trapped in the membrane is relatively large;3. If NO is only around transiently, the
membrane could act as a trap;4. There is no control over where NO goes; 5. There is no way to regulate NO release;6. There is little control over NO entry.
As you have heard today “gas channels” including aquaporin-1(AQP-1) has been
shown to transport CO2 and other gases.
Organs where AQP-1 and NO synthase are expressed
45
30
20
Gly-AQP-1
AQP-1
EC VSMC(15 g) (5 g)
AQP-1 expression by aortic EC and VSMCisolated from CD1 mice
soluble guanylatecyclase
NONOS 3
Hypothesis
AQP-1AQP-1
vascular smooth muscle cell
endothelial cell
If our hypothesis is correct:1. NO permeability (PNO) should correlate with water permeability (Pf).
2. Increasing AQP-1expression should increase NO flux.
3. Inhibitors of AQP-1 should reduce NO flux.
4. NO flux should be saturable.
5. Purified AQP-1 should transport NO.
inlet
outlet
Camera & Image Analysis
ArcLamp
objective lens
dichroic mirror
mirror
excitation 488 nm
emission535±50 nm
Measuring NO with DAF in cultured cells
Correlation of PNO and Pf in stably transfected CHO cells
1. NO permeability (PNO) correlates with water permeability (Pf).
2. Increasing AQP-1 expression should increase NO flux.
3. Inhibitors of AQP-1 should reduce NO flux.
4. NO flux should be saturable.
5. Purified AQP-1 should transport NO.
Effect of transiently transfecting CHO cells with aquaporin-1 (AQP-1) on NO influx
NO gradient by SPM (5 M NO)
1. NO permeability (PNO) correlates with water permeability (Pf).
2. Increasing AQP-1 expression increases NO flux.
3. Inhibitors of AQP-1 should reduce NO flux.
4. NO flux should be saturable.
5. Purified AQP-1 should transport NO.
Effect of DMSO, an AQP-1 inhibitor, on NO influx into transiently transfected CHO cells
NO gradient by SPM (5 M NO)
0
5
10
15
20
25
30
C AQP-1 C AQP-1
HgCl2
NO
influ
x [fl
uore
scen
ce u
nits
/sec
] p < 0.005
Effect of 20 M HgCl2, an AQP-1 inhibitor, on NO influx into transfected CHO cells
NO gradient by SPM (5 M NO)
0
10
20
30
40
50
C AQP-1 C AQP-1
DMSO
NO
influ
x[fl
uore
scen
ce u
nits
/sec
] p < 0.03
NO gradient by gas (5 M NO)
Effect of DMSO on NO influx into transiently transfected CHO cells
1. NO permeability (PNO) correlates with water permeability (Pf).
2. Increasing AQP-1 expression increases NO flux.
3. Inhibitors of AQP-1 reduce NO flux.
4. NO flux should be saturable.
5. Purified AQP-1 should transport NO.
0 1 2 3 4 5 60
10
20
30
40
50N
O in
flux
[fluo
resc
ence
uni
ts/s
ec]
NO [M]
K1/2= 0.54M
Concentration-dependent NO flux using NO gas
1. NO permeability (PNO) correlates with water permeability (Pf).
2. Increasing AQP-1 expression increases NO flux.
3. Inhibitors of AQP-1 reduce NO flux.
4. NO flux is saturable.
5. Purified AQP-1 should transport NO.
1 M NO
AQP-1
control
NO flux into proteolipisomes made with purified AQP-1
1. NO permeability (PNO) correlates with water permeability (Pf).
2. Increasing AQP-1 expression increases NO flux.
3. Inhibitors of AQP-1 reduce NO flux.
4. NO flux is saturable.
5. Purified AQP-1 increases NO transport.
Do other aquaporins transport NO?
Partial aquaporin family tree
AQP-0
AQP-5
AQP-4AQP-6
AQP-3
AQP-9
AQP-1
AQP-8
AQP-10
AQP-2
AQP-7
AQP-Z
Adapted from Agre et al. J Physiol 542:3-16, 2002
MOCK AQP-3 AQP-4
NO
influ
x [f
luor
esce
nce
units
/sec
]
0
1
2
3
4
5
6
Effect of transiently transfecting CHO cells with AQP-3 on NO influx
MOCK AQP-3 AQP-4
NO
influ
x [f
luor
esce
nce
units
/sec
]
0
1
2
3
4
5
6
Effect of transiently transfecting CHO cells with AQP-4 on NO influx
AQP-3 and AQP-4 may transport NO. More data are required.
How does NO transport by AQP-1 compare to diffusion through the bilayer in “real” cells?
Is it physiologically significant?
Aortic ring preparation
bath solution
aorticring
forcetransducer
outletinlet
gas
0
20
40
60
80
100
-10 -9 -8 -7 -6 -5
Log [Ach] concentration
% c
on
tra
ctio
n t
o P
E
WT
AQP-1 -/-
p < 0.0001
Acetylcholine-dependent relaxation of aortic rings from wild type and AQP-1 -/- mice
The reduction in Ach-induced relaxation in AQP-1 -/-mice is NOT due to:
1.Less NOS 3. There is more in AQP-1 -/- mice than WT.
2. Defective signaling down-stream of NO. Donors that release NO inside VSMCs and cGMP relax rings from AQP-1 -/- mice the same as WT.
The reduction in Ach-induced relaxation in AQP-1 -/-mice could be due to:
1. Reduced NO efflux out of endothelial cells; and/or
2. Reduced NO influx into vascular smooth muscle cells.
Stimulus: 1 mM Ach
0.0
0.5
1.0
1.5
2.0
2.5
3.0
GAPDH AQP-1 siRNA siRNA
NO
rel
ease
[pA
mps
/g]
p < 0.05
NO
rel
ease
(pA
mps
/g)
NO
rel
ease
(pA
mps
/g)
Effect of inhibiting AQP-1 on NO release by pancreatic endothelial cells
NO release by cultured aortic endothelial cells from wild type and AQP-1 -/- mice
p < 0.0001
Relaxation of denuded aortic rings to spermine NONOate, an NO donor that releases NO into the bathing media
NO influx into isolated aortic vascular smooth muscle cells from wild type and AQP-1 -/- mice
0.00
0.05
0.10
0.15
0.20
WT AQP-1
NO
influ
x [fl
uore
scen
ce u
nits
/sec
]
p < 0.002
NO
flux
[flu
ore
sce
nce
un
its/s
ec]
NO
flux
[flu
ore
sce
nce
un
its/s
ec]
We are trying to show that the reduction in NO transport by AQP-1 is physiologically relevant in vivo by showing that total peripheral resistance does not decrease in response to acetylcholine in these mice as much as wild type mice
BUT
it seems that these mice have compensation mechanism including increased prostaglandin production and NOS expression that has frustrated our attempts thus far.
Conclusion1. AQP-1 transports NO.
2. Transport of NO by AQP-1 occurs faster than by diffusion through the bilayer by about a factor of 2.
3. Transport of NO by AQP-1 appears to be physiologically significant.
4. Reduced Ach-dependent relaxation of aortic rings from AQP-1 -/- mice is due to both reduced efflux out of endothelial cells and reduced influx into vascular smooth muscle cells.