Endothelial AMP-Activated Kinase α1 Phosphorylates eNOS on Thr495 and Decreases Endothelial NO Formation

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Endothelial AMP-Activated Kinase α1

Phosphorylates eNOS on Thr495 and Decreases Endothelial NO Formation

Nina Zippel ¹, Annemarieke E. Loot ¹, Heike Stingl ^1,2,Voahanginirina Randriamboavonjy ^1,2, Ingrid Fleming^1,2 and Beate Fisslthaler^1,2,*

1 Institute for Vascular Signalling, Centre for Molecular Medicine, Johann Wolfgang Goethe University, 60590 Frankfurt, Germany; NZippel@gmx.de (N.Z.); a.loot@certe.nl (A.E.L.); Stingl@vrc.uni-frankfurt.de (H.S.); Voahangy@vrc.uni-frankfurt.de (V.R.); fleming@em.uni-frankfurt.de (I.F.)

2 DZHK (German Centre for Cardiovascular Research) partner site RhineMain, Theodor Stern Kai 7, 60590 Frankfurt, Germany

* Correspondence: fisslthaler@vrc.uni-frankfurt.de; Tel.: +49-69-6301-6994 Supplementary Materials:

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), 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.

Supplementary Figure A1. Vascular function in carotid arteries from wild-type (WT) and AMPK1^-/- mice. (A) Contraction induced by KCl (80 mmol/L), (B) 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.

-10 -9 -8 -7 -6 0

20 40 60 80 100

WT

1-/-

[ACh] (log mol/L)

Relaxation (% PE)

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20 40 60 80 100

WT

1-/-

[SNP] (log mol/L)

Relaxation (% PE)

WT 1-/- 0.0

0.5 1.0

1.5 0.1647

Force (mN/mm)

A

C D

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0 50 100

150 WT

1-/-

[PE] (log mol/L)

Contraction (%KCl)

B

looks like a significant effect – but if this is L-NAME sensitive then it’s the same as in the aorta!

Do we really need data from the carotids?

It not essential but we have it so we

may as well show it

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Figure S2. Endothelial cell specific deletion of AMPKα1. (A) AMPKα1 expression in freshly isolated pulmonary endothelial cells from AMPKα1^EC or Cre^−/− (wild-type; WT) mice. (B) Expression of eNOS, AMPKα1 and AMPKα2 in aortic ring lysates from WTor AMPKα1^EC (EC)mice. (A) The blots presented are representative of 12 additional experiments using 2 mice per group.

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 AMPKα2^EC mice (closed symbols).

(B) Relaxation curves of aortic rings to acetylcholine (ACh) after PE constriction of wild-type (open Supplementary Figure A2.Endothelial cell specific deletion of AMPK1. (A)

AMPK1 expression in freshly isolated pulmonary endothelial cells from AMPK1^ECor Cre^-/- (wild-type; WT) mice. (B) Expression of eNOS, AMPK1 and AMPK2 in aortic ring lysates from WT or AMPK1^EC (EC) mice. (A) The blots presented are representative of 12 additional experiments using 2 mice per group.

eNOS - AMPK1 - AMPK2 -

EC WT

AMPK1 -

A

B

eNOS -

b-actin -

EC WT

-55 -72 kDa

-55 -72 -130

-55 -72 kDa -36

-130

-9 -8 -7 -6 -5

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Relaxation (%PE)

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B C

Supplementary Figure A3: Effect of endothelial specific deletion of AMPK 2 on vascular reactivity of aortic rings (A) Dose dependent contraction to PE of wild-type (open symbols) or AMPK2

mice (closed symbols). (B) Relaxation curves of aortic rings to acetylcholine (ACh) after PE constriction of wild-type (open symbols) or AMPK2

mice (closed symbols).

(C) Dose-dependent relaxation to SNP. The graphs summarize data obtained from 6 animals

in each group.

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symbols) or AMPKα2^EC mice (closed symbols). (C) Dose-dependent relaxation to SNP. The graphs summarize data obtained from 6 animals in each group.

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 AMPKα1^EC (α1^EC) mice; n = 6 animals in each group. (C,D) Time- dependent 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 AMPKα1^EC (α1^EC) mice; n = 4 animals in each group. (E) Effects of the AMPK activators on the phosphorylation of AMPK (on Thr172) and ACC (Ser79) 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 min. Comparable results were obtained in 3 additional independent experiments.

Supplementary Figure A4.

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 AMPK1

(1EC) mice; n=6 animals in each group. (C&D) Time-dependent 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 AMPK1

(1EC) mice; n=4 animals in each group. (E) Effects of the AMPK activators on the phosphorylation of AMPK (on Thr172) and ACC (on

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.

PT-1 PT-1

- pAMPK -

AICAR Resv Basal Basal 991 991

10 60 10 60 min

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1E C + L - N A M E [A m u re n s in G ] (µ g /m l)

Relaxation (%PE)

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C D

E ^{Basal 991 AICAR Basal}

-pACC

-pAMPK

-b-actin

PT-1