1. AMPK Dilates Resistance Arteries via Activation of SERCA and BKCa Channels in Smooth MuscleNovelty and Significance
by Holger Schneider, Kai Michael Schubert, Stephanie Blodow, Claus-Peter Kreutz, Serap Erdogmus, Margarethe Wiedenmann, Jiehua Qiu, Theres Fey, Peter Ruth, Lubomir T. Lubomirov, Gabriele Pfitzer, Michael Mederos y Schnitzler, D. Grahame Hardie, Thomas Gudermann, and Ulrich Pohl
Hypertension
Volume 66(1):
June 10, 2015
Copyright © American Heart Association, Inc. All rights reserved.

2. 5′-AMP–activated protein kinase (AMPK)-mediated, endothelium-independent vasodilation paralleled by a decrease in [Ca2+]i.
5′-AMP–activated protein kinase (AMPK)-mediated, endothelium-independent vasodilation paralleled by a decrease in [Ca2+]i. Representative example of an original recording of the effects of A (A76, 1–100 µmol/L, gray) versus time control (black) on diameter (A) and intracellular calcium (B). Arrowheads indicate application of A76 at increasing concentrations or sham solution. The small resistance artery preconstricted with 0.3 μmol/L norepinephrine (NE) shows potent vasodilation associated with a decrease of [Ca2+]i only when AMPK was stimulated. The application of the CaV-channel blocker nifedipine (5 μmol/L) elicited no further dilation and only minor additional calcium decrease. Microvessels showed dose-dependent and endothelium-independent vasodilation (C) associated with a decrease of [Ca2+]i (D) when exposed to A76 in concentrations reaching from 10–6 to 3×10–4 mol/L (n=4–5). Note the stable calcium signal of the time controls in comparison with the decrease in the AMPK-stimulated vessels. *P<0.05, intact endothelium vs without endothelium, 2-way ANOVA, Tukey.
Holger Schneider et al. Hypertension. 2015;66:
Copyright © American Heart Association, Inc. All rights reserved.

3. 5′-AMP–activated protein kinase (AMPK) effects in vascular smooth muscle cell (VSMC) involve activation of BKCa channels and associated hyperpolarization.
5′-AMP–activated protein kinase (AMPK) effects in vascular smooth muscle cell (VSMC) involve activation of BKCa channels and associated hyperpolarization. A (top row), Patch clamp studies in freshly isolated smooth muscle cells derived from small mouse mesenteric arteries demonstrating BKCa-dependent outward currents on stimulation with A76 in wild-type (WT) but not in BKCa−/− mice (B) and increased current densities on the 2 independent AMPK stimulators A76 and PT1 in WT but not −/− cells (C; n=3–4, *P<0.05; t test). D, Membrane potential recordings obtained in smooth muscle cells of mouse mesenteric arteries in situ under nonstimulated conditions (resting membrane potential, RMP), on stimulation by norepinephrine (NE) alone, together with A76 in the presence or absence of iberiotoxin (IbTx) and thapsigargin (TG; n=13–25 measurements from 5–13 vessels, *P<0.05, 1-way ANOVA, Holm–Sidak). Bottom row, Results obtained in isolated smooth muscle cells freshly obtained from hamster vessels. E, BKCa-dependent outward currents in the absence and presence (F) of A76 at increasing holding potentials. G, Dose dependency of A76 induced outward currents and effects of compound C and BKCa channel blockers (H). J, Membrane potentials of isolated cells showing hyperpolarization induced by A76 and reversible inhibition by the AMPK inhibitor compound C (n=5–27, **P<0.01, ***P<0.001; paired t test).
Holger Schneider et al. Hypertension. 2015;66:
Copyright © American Heart Association, Inc. All rights reserved.

4. Reduced 5′-AMP–activated protein kinase (AMPK) effects under sarcoplasmic/endoplasmic Ca2+-ATPase (SERCA) inhibition.
Reduced 5′-AMP–activated protein kinase (AMPK) effects under sarcoplasmic/endoplasmic Ca2+-ATPase (SERCA) inhibition. Thapsigargin and iberiotoxin show inhibitory effects on the vasodilation (A) and calcium decrease (B) mediated by AMPK. The single blockade of BKCa channels with iberiotoxin (IbTx, 100 nmol/L) caused no prominent reduction of the AMPK-induced vasodilator effects, contrary to SERCA inhibition with thapsigargin (TG, 1 µmol/L). Simultaneous application of IbTx and TG virtually abolished both the A76-induced vasodilation and the calcium decrease (n=4–6, *P<0.05, 2-way ANOVA, Tukey). n.s. indicates no significant difference.
Holger Schneider et al. Hypertension. 2015;66:
Copyright © American Heart Association, Inc. All rights reserved.

5. A, Increased sarcoplasmic/endoplasmic Ca2+-ATPase activity on 5′-AMP–activated protein kinase (AMPK) stimulation.
A, Increased sarcoplasmic/endoplasmic Ca2+-ATPase activity on 5′-AMP–activated protein kinase (AMPK) stimulation. A, Stimulation of calcium release via caffeine (10 mmol/L) under calcium-free conditions elicited a smaller calcium transient in the time control vessels (dark triangles) compared with AMPK-activated vessels (gray circles). B, In hamster arteries, as well as in mouse arteries (C), 1 mmol/L caffeine and 10 mmol/L caffeine, respectively, induced higher transients after A76 pretreatment than control treatment. This transient could be inhibited by thapsigargin (TG, B). There was no statistically significant difference between the native state (time control) and thapsigargin, albeit the tendency suggests an intermediate store filling (B: n=5–9, *P<0.05, Kruskal–Wallis test, Dunn’s method; C: n=4 each,*P<0.05, t test).
Holger Schneider et al. Hypertension. 2015;66:
Copyright © American Heart Association, Inc. All rights reserved.

6. Increased phosphorylation of the sarcoplasmic/endoplasmic Ca2+-ATPase modulator phospholamban in vascular smooth muscle after 5′-AMP–activated protein kinase stimulation.
Increased phosphorylation of the sarcoplasmic/endoplasmic Ca2+-ATPase modulator phospholamban in vascular smooth muscle after 5′-AMP–activated protein kinase stimulation. A, Representative Western blot from pooled femoral arteries to assess phosphorylation of phospholamban at threonine 17 (pT17-PLB, 24 kDa) in pooled arteries stimulated with norepinephrine and treated either with 30 µmol/L A76 (A76) or with solvent only (0.03 ‰ dimethyl sulfoxide, Ctrl). A76 increased PLB phosphorylation. Heart tissue (Heart) stimulated with isoprenaline (ISO) was used as a positive control. B, T17 phosphorylation in A76-stimulated arteries was tripled in comparison with vehicle-treated control (n=4, *P<0.05, paired t test). TG indicates thapsigargin.
Holger Schneider et al. Hypertension. 2015;66:
Copyright © American Heart Association, Inc. All rights reserved.

7. Proposed model of 5′-AMP–activated protein kinase (AMPK)-mediated effects in vascular smooth muscle cell (VSMC).
Proposed model of 5′-AMP–activated protein kinase (AMPK)-mediated effects in vascular smooth muscle cell (VSMC). AMPK activates BKCa channels leading to hyperpolarization and (partial) closure of CaV channels. In addition, AMPK increases sarcoplasmic/endoplasmic Ca2+-ATPase (SERCA) activity involving phospholamban (PLB) phosphorylation. Both mechanisms result in a reduced [Ca2+]i which ultimately leads to relaxation of VSMC. Although both mechanisms are activated, SERCA stimulation alone already is sufficient for the AMPK-mediated [Ca2+]i reduction and vasodilation.
Holger Schneider et al. Hypertension. 2015;66:
Copyright © American Heart Association, Inc. All rights reserved.