looks like a significant effect – but if this
EndothelialisAMP-Activated α1 L-NAME sensitive thenKinase it’s the same as in the aorta! on Thr495 and Decreases Phosphorylates eNOS we really need data from the EndothelialDoNO Formation
carotids? 1,2, Voahanginirina It not essential but Stingl we have it so we Randriamboavonjy 1,2, Nina Zippel 1, Annemarieke E. Loot 1, Heike 1,2,* Ingrid Fleming1,2 and Beateas Fisslthaler may well show it Institute for Vascular Signalling, Centre for Molecular Medicine, Johann Wolfgang Goethe University, 60590 Frankfurt, Germany;
[email protected] (N.Z.);
[email protected] (A.E.L.);
[email protected] (H.S.);
[email protected] (V.R.);
[email protected] (I.F.) 2 DZHK (German Centre for Cardiovascular Research) partner site RhineMain, Theodor Stern Kai 7, 60590 Frankfurt, Germany * Correspondence:
[email protected]; Tel.: +49-69-6301-6994 1
Supplementary Materials:
A
B 150
0.1647
Contraction (%KCl)
Force (mN/mm)
1.5
1.0
0.5
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0 0.0 WT
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[ACh] (log mol/L) -10 -9 -8 -7 -6
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[SNP] (log mol/L) -9 -8 -7 -6
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Relaxation (% PE)
Relaxation (% PE)
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-8 -7 -6 [PE] (log mol/L)
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WT 1-/-
Figure S1. Vascular function in carotid arteries from wild-type (WT) and AMPKα1−/− mice. (A) Contraction induced by KCl (80 mmol/L), (B) concentration response curves to phenylephrine (PE), Supplementary Figure A1. Vascular function in carotid arteries from wild-type and relaxation to (C) acetylcholine (ACh) or (D) sodium nitroprusside (SNP) in PE-contracted -/- mice. (A) (WT) curves and AMPK1 Contraction induced by KCl (80 mmol/L), (B) vessels. The graphs summarize data obtained from 7 animals in each group. concentration response curves to phenylephrine (PE), and relaxation curves to
(C) acetylcholine (ACh) or (D) sodium nitroprusside (SNP) in PE-contracted vessels. The graphs summarize data obtained from 7 animals in each group.
1
EC
A
WT EC
WT kDa
eNOS -
-130 -72
AMPK1 -
-55
b-actin -
-36 EC
WT
B
kDa
eNOS -
-130 -72
AMPK1 -
-55 -72
AMPK2 -
-55
Figure S2. Endothelial cell specific deletion of AMPKα1. (A) AMPKα1 expression in freshly isolated pulmonary endothelial cells from AMPKα1EC or Cre−/− (wild-type; WT) mice. (B) Expression of eNOS, AMPKα1 and AMPKα2 in aortic ring lysates from WT or AMPKα1EC (EC) mice. (A) The blots presented are representative of 12 additional experiments using 2 mice per group.
Supplementary Figure A2. Endothelial cell specific deletion of AMPK1. (A) AMPK1 expression in freshly isolated pulmonary endothelial cells from AMPK1EC or Cre-/- (wild-type; WT) mice. (B) Expression of eNOS, AMPK1 and AMPK2 in aortic ring lysates from WT or AMPK1EC (EC) mice. (A) The blotsApresented are representative of 12 additional experiments using 2 mice per group. WT 1 .0
F o rc e (g )
2EC
0 .5
0 .0 -9
-8
-7
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[P E ] ( lo g m o l/L )
B
C
[A C h ] ( lo g m o l/L ) -9
-8
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-6
-1 0
40 60 WT 2EC
R e la x a tio n (% P E )
R e la x a tio n (% P E )
20
100
-9
-8
-7
-6
0
0
80
[S N P ] ( lo g m o l/L )
-5
20 40 60 80 100
WT 2EC
Figure S3. Effect of endothelial specific deletion of AMPKα2 on vascular reactivity of aortic rings (A) Dose dependent contraction to PE of wild-type (open symbols) or AMPKα2EC mice (closed symbols). (B) Relaxation curves of aortic rings to acetylcholine (ACh) after PE constriction of wild-type (open
2 Supplementary Figure A3: Effect of endothelial specific deletion of AMPK2 on vascula reactivity of aortic rings (A) Dose dependent contraction to PE of wild-type (open symbols) o AMPK2EC mice (closed symbols). (B) Relaxation curves of aortic rings to acetylcholin (ACh) after PE constriction of wild-type (open symbols) or AMPK2EC mice (closed symbols
symbols) or AMPKα2EC mice (closed symbols). (C) Dose-dependent relaxation to SNP. The graphs summarize data obtained from 6 animals in each group.
[R e s v e ra tro l] (µ m o l/L )
A
0
50
100
0
WT 1E C -5 0
W T + L -N A M E 1 E C + L -N A M E
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1 E C + L -N A M E
T im e ( m in )
D
60
10
1EC W T + L -N A M E -1 0 0
T im e ( m in )
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5
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R e la x a tio n (% P E )
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[A m u re n s in G ] ( µ g /m l)
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W T -S O L 1EC W T + L -N A M E
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E
R e la x a tio n (% P E )
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1 E C + L -N A M E
Basal 991 AICAR
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W T -S O L -5 0
W T + L -N A M E
-1 0 0
Basal
1EC 1 E C + L -N A M E
PT-1 -pACC -pAMPK
-b-actin
Figure S4. Effect of AMPK activators on the relaxation of aortic rings. (A,B) Concentration dependent effects of resveratrol (A) and amurensin G (B) on vascular tone in phenylephrine preconstricted aortic rings from wild-type (WT) and AMPKα1EC (α1EC) mice; n = 6 animals in each group. (C,D) Timedependent effects of PT-1 (C, 30 µmol/L) and 991 (D; 30 µmol/L) on vascular tone in phenylephrine preconstricted aortic rings from wild-type (WT) and AMPKα1EC (α1EC) mice; n = 4 animals in each Supplementary Figure A4. Effect of AMPK activators on the relaxation of aortic rings. group. (E) Effects of the AMPK activators on the phosphorylation of AMPK (on Thr172) and ACC (A&B) Concentration dependent effects of resveratrol (A) and amurensin G (B) on vascular (Ser79) in endothelial cells isolated from aortic aortic rings from mice. Experiments were tone in phenylephrine preconstricted ringswild-type from wild-type (WT) and AMPK1EC performed in the absence (Basal) and presence of 991 (30 µmol/L), AICAR (0.5 mmol/L) or PT-1 (30 (1EC) mice; n=6 animals in each group. (C&D) Time-dependent effects of PT-1 (C, 30 µmol/L) for 60 min. were in tone 3 additional independent preconstricted experiments. µmol/L) and Comparable 991 (D; 30 results µmol/L) onobtained vascular in phenylephrine aortic rings
from wild-type (WT) and AMPK1EC (1EC) mice; n=4 animals in each group. (E) Effects of the AMPK activators on the phosphorylation of AMPK (on Thr172) and ACC (on ???) in endothelial cells isolated from aortic rings from wild-type mice. experiments were performed in the absence (Basal) and presence of 991 (30 µmol/L), AICAR (0.5 mmol/L) or PT-1 (30 µmol/L) for 60 minutes. Comparable results were obtained in 3 additional independent experiments. 3
