AMP-activated Protein Kinase Phosphorylates and Desensitizes Smooth Muscle Myosin Light Chain Kinase*♦

Smooth muscle contraction is initiated by a rise in intracellular calcium, leading to activation of smooth muscle myosin light chain kinase (MLCK) via calcium/calmodulin (CaM). Activated MLCK then phosphorylates the regulatory myosin light chains, triggering cross-bridge cycling and contraction. Here, we show that MLCK is a substrate of AMP-activated protein kinase (AMPK). The phosphorylation site in chicken MLCK was identified by mass spectrometry to be located in the CaM-binding domain at Ser815. Phosphorylation by AMPK desensitized MLCK by increasing the concentration of CaM required for half-maximal activation. In primary cultures of rat aortic smooth muscle cells, vasoconstrictors activated AMPK in a calcium-dependent manner via CaM-dependent protein kinase kinase-β, a known upstream kinase of AMPK. Indeed, vasoconstrictor-induced AMPK activation was abrogated by the STO-609 CaM-dependent protein kinase kinase-β inhibitor. Myosin light chain phosphorylation was increased under these conditions, suggesting that contraction would be potentiated by ablation of AMPK. Indeed, in aortic rings from mice in which α1, the major catalytic subunit isoform in arterial smooth muscle, had been deleted, KCl- or phenylephrine-induced contraction was increased. The findings suggest that AMPK attenuates contraction by phosphorylating and inactivating MLCK. This might contribute to reduced ATP turnover in the tonic phase of smooth muscle contraction.


Smooth muscle contraction is initiated by a rise in intracellular calcium, leading to activation of smooth muscle myosin light chain kinase (MLCK) via calcium/calmodulin (CaM). Activated MLCK then phosphorylates the regulatory myosin light chains, triggering cross-bridge cycling and contraction. Here, we show that MLCK is a substrate of AMP-activated protein kinase (AMPK). The phosphorylation site in chicken MLCK was identified by mass spectrometry to be located in the CaM-binding domain at Ser 815 . Phosphorylation by AMPK desensitized MLCK by increasing the concentration of CaM required for half-maximal activation. In primary cultures of rat aortic smooth muscle cells, vasoconstrictors activated AMPK in a calcium-dependent manner via
CaM-dependent protein kinase kinase-␤, a known upstream kinase of AMPK. Indeed, vasoconstrictor-induced AMPK activation was abrogated by the STO-609 CaM-dependent protein kinase kinase-␤ inhibitor. Myosin light chain phosphorylation was increased under these conditions, suggesting that contraction would be potentiated by ablation of AMPK. Indeed, in aortic rings from mice in which ␣1, the major catalytic subunit isoform in arterial smooth muscle, had been deleted, KCl-or phenylephrine-induced contraction was increased. The findings suggest that AMPK attenuates contraction by phosphorylating and inactivating MLCK. This might contribute to reduced ATP turnover in the tonic phase of smooth muscle contraction.
Vascular smooth muscle contraction can be initiated by agonists acting via G protein-coupled receptors (1), resulting in phospholipase C␤ activation and the formation of inositol 1,4,5-trisphosphate and diacylglycerol. Inositol 1,4,5trisphosphate induces Ca 2ϩ release from sarcoplasmic reticulum stores (2), whereas diacylglycerol activates protein kinase C (3). The rise in [Ca 2ϩ ] i is further increased by Ca 2ϩ influx through voltage-gated Ca 2ϩ channels. Binding of Ca 2ϩ to calmodulin (CaM) 5 (4) activates smooth muscle myosin light chain kinase (smMLCK) by inducing a conformational change that removes an autoinhibitory domain from the kinase active site and allows smMLCK to phosphorylate 20-kDa regulatory myosin light chains (MLC) at Ser 19 (5). MLC phosphorylation is necessary and sufficient for the initiation of contraction, triggering cross-bridge formation and contraction (6).
The activity of smMLCK itself can be modulated by phosphorylation, which changes its V max or affinity for Ca 2ϩ /CaM. smMLCK is phosphorylated at two sites (7,8) by cAMP-dependent protein kinase (PKA). One site (site A, corresponding to Ser 815 in chicken and Ser 992 in rabbit smMLCK and conserved in the human, mouse, and rat sequences) lies in the C-terminal CaM-binding domain. Treatment with PKA inactivates MLCK by decreasing its affinity for Ca 2ϩ /CaM (9). Similar changes in affinity have also been observed after phosphorylation by Ca 2ϩ /CaM-dependent kinase II (7) and protein kinase C (10). Moreover, smMLCK contains several consensus sequences for proline-directed kinases, and phosphorylation by MAPK increases the V max with no change in affinity for Ca 2ϩ / CaM (10,11). AMP-activated protein kinase (AMPK) acts as an energy sensor at both the cellular and systemic levels in mammals (12,13). AMPK is a heterotrimer consisting of a catalytic ␣-subunit and two regulatory subunits, ␤ and ␥. Each subunit has multiple isoforms (␣1, ␣2, ␤1, ␤2, ␥1, ␥2, and ␥3), giving 12 possible combinations of holoenzyme with different tissue distribution and subcellular localization. AMPK can be activated by changes in the intracellular AMP/ATP ratio, as occurs, for example, under anoxia or other stresses (12), or via a rise in [Ca 2ϩ ] i (13,14). Activation by the upstream kinases LKB1 (Peutz-Jeghers protein) and Ca 2ϩ /CaM-dependent protein kinase kinase-␤ (CaMKK␤) occurs via phosphorylation of Thr 172 in the activation loop of the catalytic ␣-subunits (13,14). AMP not only allosterically stimulates AMPK, but also protects Thr 172 against dephosphorylation by protein phosphatases (15). Therefore, changes in both intracellular Ca 2ϩ and AMP concentrations could play separate or interdependent roles in the regulation of AMPK activity. Once activated, AMPK decreases ATP consumption and stimulates ATP-producing processes (12).
In this work, we show that smMLCK is a substrate of AMPK. Phosphorylation by AMPK decreased the affinity of smMLCK for Ca 2ϩ /CaM. We also show that stimulation of aortic smooth muscle cells by vasoconstrictors led to Ca 2ϩ -dependent AMPK activation, which was blocked by the CaMKK␤ inhibitor. Moreover, in aortic rings of ␣1-AMPK knock-out mice, KCl-and phenylephrine-induced contraction was potentiated. The findings support the emerging notion that the role of AMPK extends beyond that of energy sensing.
To study the effects of phosphorylation by AMPK and PKA on MLCK activity, chicken gizzard smMLCK (0.4 g) was incubated in phosphorylation buffer with 0.1 mM MgATP in a final volume of 40 l with no further additions or with AMPK (60 milliunits) or PKA (50 milliunits). After 10 min at 30°C, the reaction mixtures were placed on ice and diluted 5-fold in phosphorylation buffer. Aliquots (2.5 l) were assayed for MLCK in a final volume of 20 l of phosphorylation buffer containing 10 g/ml CaM, 0.1 mg/ml purified chicken gizzard MLC, 6 mM CaCl 2 , 5 mM EGTA, and 0.1 mM [␥-32 P]MgATP (specific radioactivity, 500 cpm/pmol). After 4 min at 30°C, aliquots (10 l) were spotted on 1 ϫ 1-cm squares of Whatman P-81 phosphocellulose paper and immersed in 75 mM phosphoric acid. After washing four times with phosphoric acid and once with acetone, the papers were dried and counted by Č erenkov radiation.
AMPK was assayed in polyethylene glycol fractions (22,23) or in anti-␣1/␣2-AMPK antibody immunoprecipitates of cell lysates (16,20). Purified recombinant activated AMPK (22) and purified PKA catalytic subunits (23) were assayed as indicated. One unit of protein kinase activity corresponds to the amount of enzyme that catalyzes the incorporation of 1 nmol of 32 P/min into its substrate under the assay conditions. Phosphorylation Site Identification by Mass Spectrometry-The phosphorylated band corresponding to MLCK phosphorylated in vitro by AMPK (as described above) was cut from Coomassie Blue-stained gels and digested with 1 g of sequencing grade trypsin as described (24). Peptides were separated by reversephase narrow-bore HPLC at a flow rate of 200 l/min, and radioactive peaks were analyzed by nanoelectrospray ionization tandem mass spectrometry in an LCQ Deca XP Plus ion-trap mass spectrometer (Thermo Finnigan, San Jose, CA).
Pulldown Experiments with CaM-Agarose-smMLCK from chicken gizzard (0.5 g) was incubated in phosphorylation buffer with 0.1 mM MgATP in a final volume of 50 l with no other additions or with AMPK (60 milliunits) for 60 min at 30°C. The reaction was incubated with CaM-agarose (20 l of a 1:1 suspension) in the same buffer supplemented with 5 mM CaCl 2 for 60 min at 30°C. Following centrifugation, the pellet was boiled in SDS-PAGE sample buffer and analyzed by immunoblotting with anti-full-length MLCK as primary antibody. The same procedure was followed for centrifuged aortic smooth muscle cell (ASMC) extracts (150 l).
Isolation of Aortic Explants and Treatment and Lysis of ASMCs-Primary rat ASMC cultures were prepared from aortic explants. Thoracic aortas from male Wistar rats were rapidly removed, cleaned of any adherent connective tissue, opened longitudinally, and rubbed along the inner surface to remove the endothelium. Aortas were then cut into squares (ϳ10 mm 2 ) and placed in 6-well microtiter plates in Dulbecco's modified Eagle's medium supplemented with 10% (w/v) fetal bovine serum, 4 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. After 7-10 days, when suitable amounts of smooth muscle cells growing out of the explant were visible, explants were removed, and the cells were harvested in a tissue culture flask (175-cm 2 growth area). When they had reached confluence, the cells were passaged once before the experiment. The medium of early-passage ASMCs was replaced 24 h prior to treatment with Dulbecco's modified Eagle's medium without fetal bovine serum and containing a lower glucose concentration (1 g/liter). Cells were incubated with angiotensin II, vasopressin, or endothelin-1 at 10 Ϫ7 M with or without a 60-min pretreatment with the CaMKK␤ inhibitor STO-609 (10 M) for the times indicated in the figures and legends. Control cells received an appropriate volume of relevant vehicle. For AMPK assays on immunoprecipitates and immunoblotting, cells were lysed in buffer containing 50 mM HEPES (pH 7.6), 50 mM KCl, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1% (w/v) Triton X-100, 5 mM ␤-glycerophosphate, and a mixture of protease inhibitors (Roche Applied Science). Lysates were centrifuged at 13,000 ϫ g for 10 min, and protein concentrations in the resulting supernatants were determined. For immunoblotting MLC, cells were extracted in 8 M urea, 2% (w/v) Nonidet P-40, 15 mM ␤-mercaptoethanol, and a mixture of protease inhibitors.
Immunoblotting-Extracts were prepared as described above. Proteins were resolved by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes (Millipore). The membranes were probed with the following antibodies: antiphospho-Thr 172 AMPK, anti-phospho-Ser 221 ACC2, antiphospho-Thr 202 /Tyr 204 ERK1/2, anti-phospho-Thr 180 /Tyr 182 p38 MAPK, anti-phospho-Ser 19 MLC, and anti-full-length smMLCK, AMPK, and eukaryotic elongation factor-2. The membranes were incubated either in Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE) for detection and quantification with the Odyssey infrared imager (LI-COR Biosciences) or in 5% nonfat milk for detection by enhanced chemiluminescence. Band intensities were quantified by scanning films and processing the image intensities with the program Image J (133 for Mac OS X).
Measurement of Tension Development in Aortic Rings-Animal experiments were approved by the Ethical Committee of the Faculty of Medicine, Université catholique de Louvain. Wild-type and ␣1-AMPK knock-out mice had free access to water and food. Male mice (4 months old) were anesthetized with diethyl ether and killed by decapitation. Aortas were rapidly removed, immersed in Krebs bicarbonate buffer (118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO 3 , 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 2.5 mM CaCl 2 , and 5 mM glucose), gassed with a mixture of 95% O 2 and 5% CO 2 , and carefully cleaned of all fat and connective tissue. Aortic rings (Ϯ2 mm) were dissected and mounted between two hooks under a tension of 20 millinewtons in a 3-ml cuvette continuously perfused with Krebs solution supplemented with N G -nitro-L-arginine (100 M) at 37°C (25). Muscle tone was measured using an isometric force transducer (25). The artery segment was incubated for 30 min in Krebs solution and thereafter stimulated with a KCl solution or with phenylephrine at concentrations ranging from 10 Ϫ9 to 10 Ϫ5 M. The KCl-depolarizing solutions were obtained by iso-tonic replacement of NaCl with KCl in Krebs solution (26). Tension developed was expressed as millinewtons/mm. Each experiment was repeated on at least 10 different aortic rings and on five mice. For calcium measurements, aortic rings (Ϯ2 mm) were dissected and maintained for 3-3.5 h at room temperature in Krebs bicarbonate buffer containing 5 M fura-2 acetoxymethyl ester and 0.05% Cremophor EL. The calcium signal was measured in a JASCO CAF fluorometer.
Other Methods-Protein concentration was estimated by the method of Bradford (27) with bovine serum albumin as a standard.
Statistical Analyses-Data are expressed as the means Ϯ S.E. Student's two-sided t test or one-way analysis of variance (Bonferroni adjustment) was used to assess the statistical significance (p Ͻ 0.05) of the data.

RESULTS
In Vitro Experiments-smMLCK was identified serendipitously as a potential AMPK target by mass spectrometric analysis of liver proteins eluting from a CaM-Sepharose column and containing a M r 120,000 gel band phosphorylated in vitro by AMPK. We therefore investigated whether smMLCK might be a bona fide AMPK substrate and whether phosphorylation affected its activity. Upon incubation with [␥-32 P]Mg-ATP and activated AMPK or PKA catalytic subunits, purified chicken gizzard smMLCK was phosphorylated to a stoichiometry of 0.35 or 0.75 mol of phosphate incorporated per mol of smMLCK protein, respectively, after 20 min of incubation. In both cases, phosphorylation was reduced by Ca 2ϩ /CaM (Fig.  1A), suggesting that the phosphorylation site(s) lie in the C-terminal CaM-binding domain. Indeed, phosphorylation by both PKA and AMPK increased the K 0.5 for CaM (Fig. 1B) from 0.04 g/ml in the control to 0.17 in the PKA-and AMPK-treated samples (p Ͻ 0.05, n ϭ 4).
After maximal phosphorylation by AMPK/[␥-32 P]MgATP and SDS-PAGE, the smMLCK band was digested with trypsin; peptides were separated by reverse-phase HPLC; and the major radioactive peak ( Fig. 2A) was analyzed by mass spectrometry. Ser 815 was identified as the phosphorylated residue in the phosphorylated peptide ( 813 LSSMAMISGMSGR 825 ). Ser 815 corresponds to site A in the C-terminal CaM-binding domain of smMLCK (Fig. 2B), known to be phosphorylated by PKA (9), Ca 2ϩ /CaM-dependent kinase II (7), and protein kinase C (28). The lower stoichiometry of phosphorylation of smMLCK by AMPK compared with PKA (Fig. 1A) is explained by the fact that AMPK phosphorylates a single site, whereas PKA is known to phosphorylate two sites in smMLCK.
We further confirmed the lower affinity for CaM of phosphorylated smMLCK in pulldown experiments. smMLCK treated with ATP with or without AMPK was incubated with CaM-agarose, and the pellet obtained after centrifugation was immunoblotted with anti-full-length smMLCK antibody. smMLCK was not detected in the pellet after phosphorylation by AMPK, whereas it bound CaM-agarose in the control condition (Fig. 1C).
Experiments in ASMCs-AMPK activation by vasoconstrictors was tested in primary cultures of ASMCs generated from rat aortic explants. AMPK was immunoprecipitated with anti-␣1and anti-␣2-AMPK antibodies prior to AMPK assay. The AMPK catalytic ␣1-subunit isoform accounted for 90% of the total AMPK activity in ASMC lysates (data not shown), confirming results obtained with porcine carotid arteries (29). Treatment with 10 Ϫ7 M vasopressin, angiotensin II, or endothelin-1 led to AMPK activation (Fig. 3A). Phenylephrine treatment of ASMCs did not lead to AMPK activation, presumably because of the disappearance of ␣1-adrenergic receptors in these cultured cells. Because Ca 2ϩ mediates vasoconstrictor action, AMPK activation was measured in ASMCs incubated with Ca 2ϩ ionophore. When measured in polyethylene glycol fractions of cell lysates, AMPK was activated ϳ2-fold after 2 min of treatment with ionophore (Fig. 3B). It is noteworthy that at this concentration of A23187, adenine nucleotide concentrations were shown previously to be unmodified (30), suggesting that AMPK is probably not activated via a rise in [AMP] through the LKB1 pathway. Moreover, AMPK activation induced by vasopressin was blocked by 5 mM EGTA in the incubation medium (Fig. 3C).
An obvious candidate for mediating vasoconstrictor-induced AMPK activation in ASMCs is CaMKK␤, an upstream kinase in the AMPK cascade (13,14). The CaMKK␤ gene codes for several splice variants. By probing with a polyclonal antibody that recognizes CaMKK␤, ASMC extracts were shown to contain two immunoreactive species (Fig. 4): a major band (M r 65,000) corresponding to the CaMKK␤1 isoform predominantly expressed in normal rat brain and a minor band (M r 60,000) corresponding to the CaMKK␤3 isoform predominantly expressed in HeLa cells (31).
STO-609 is a relatively selective and cell-permeable inhibitor of CaMKK␤ (32). At a concentration of 10 M, STO-609 completely blocked AMPK activation by vasopressin, angiotensin II, and endothelin-1 (Fig. 3A). Immunoblotting of ASMC lysates indicated that the effect of STO-609 on AMPK activity was due to a decrease in Thr 172 AMPK ␣-subunit phosphorylation, with no change in total AMPK ␣-subunit expression (Fig. 3A).
Because the MAPK pathway is known to be activated in ASMCs by agonists acting via G protein-coupled receptors (33), we examined ERK1/2 and p38 MAPK activation in comparison with AMPK activation in response to vasopressin by immunoblotting with activation loop phospho-specific antibodies (supplemental Fig. 1). Phosphorylation of these protein kinases was transient, with maximal phosphorylation of ERK and p38 MAPK at 5 min preceding that of AMPK and its substrate ACC2 (supplemental Fig. 1). To test the potential involvement of the CaMKK␤/AMPK and MAPK cascades in MLC phosphorylation, ASMCs were treated with vasopressin in the presence or absence of STO-609, PD98059, or SB203580, inhibitors of CaMKK␤, ERK1/2, and p38 MAPK, respectively. As expected, incubation with vasopressin led to an increase in MLC Ser 19 phosphorylation (Fig. 5), which was significant after 30 min of treatment. Preincubation with STO-609 accelerated MLC phosphorylation, which became maximal after 5 min of treatment with vasoconstrictor (Fig. 5). By contrast, abrogation of ERK1/2 and p38 MAPK activation with PD98059 and SB203580, respectively, had no effect on the time course of MLC phosphorylation (data not shown), suggesting that antagonism of the CaMKK␤/AMPK axis is solely responsible for the rise in MLC phosphorylation in response to vasopressin treatment. A decreased affinity of smMLCK for CaM induced by vasopressin treatment was demonstrated using CaM-agarose pulldown experiments and analysis of the pellet by immunoblotting with anti-full-length smMLCK as primary antibody (Fig. 6). The smMLCK signal decreased in extracts from vasopressin-treated ASMCs, but this effect was absent after prein- cubation with STO-609 (Fig. 6), suggesting that the CaMKK␤/ AMPK axis is mediating the decreased affinity of smMLCK for CaM induced by vasopressin.
Experiments in ␣1-AMPK Knock-out Mice-As the ␣1-subunit isoform accounted for 90% of the total AMPK in vascular smooth muscle, we used ␣1-AMPK knock-out mice (34). The major phenotypic change observed in ␣1-AMPK knock-out mice is splenomegaly (35), but there were no differences in the concentrations of adrenaline, noradrenaline, or dopamine in urine compared with control mice. 6 The functional responses of aorta induced to contract by depolarization with KCl (10 rings/five mice) or by treatment with phenylephrine (14 rings/ seven mice) were studied (Fig. 7). Because AMPK is known to phosphorylate endothelial nitric-oxide synthase, the rings were treated with the nitric-oxide synthase inhibitor N G -nitro-L-arginine. The maximal extent of force developed in aortic rings in response to increasing concentrations of phenylephrine was significantly greater in the ␣1-AMPK knock-out mice than in the wild-type controls (Fig. 7A), the sensitivity to phenylephrine being the same, however, in both groups. The increased contraction observed in ␣1-AMPK knock-out mice was also seen in rings depolarized with increasing concentrations of KCl (Fig. 7B). In addition, the increase in Ca 2ϩ induced by phenylephrine treatment (Fig. 7C) or by KCl-induced depolarization (Fig. 7D) produced a greater increase in tension development in rings from knockout compared with wild-type mice, and this increased contractility in the ␣1-AMPK knock-out mice persisted in the presence of the calcium ionophore ionomycin (data not shown). These findings further support the contention that AMPK activation in vascular smooth muscle imparts a brake on contraction.
The effect of KCl-induced depolarization on AMPK activity was assessed in ASMCs by monitoring ACC2 phosphorylation. An increase in the extent of phosphorylation of ACC2 was seen after 5 min of treatment with KCl solution (143 mM KCl, 1.2 mM CaCl 2 , 1.2 mM MgCl 2 , 11.6 mM HEPES, and 11.5 mM glucose) (supplemental Fig. 2). Moreover, ACC2 phosphorylation was prevented when the cells were preincubated with STO-609. However, phenylephrine was without effect in ASMCs, as neither AMPK activation nor MLC phosphorylation was seen with this agonist (data not shown).

DISCUSSION
The conclusion that AMPK participates in the control of smooth muscle contraction is based on the following: (i) vasoconstrictors activated AMPK in ASMCs; (ii) AMPK phosphorylated smMLCK and reduced its affinity for CaM; (iii) inhibition of CaMKK␤ antagonized vasoconstrictor-induced AMPK activation and smMLCK desensitization and resulted in enhanced MLC phosphorylation; and (iv) aortic contractility due to KCl-induced depolarization or phenylephrine treatment was increased in ␣1-AMPK knock-out mice. Together, the data suggest that the CaMKK␤/AMPK axis modulates Ca 2ϩ -dependent contraction via AMPK-induced phosphorylation of smMLCK.
Ser 815 was identified as the phosphorylation site for AMPK in smMLCK. The sequence surrounding Ser 815 of chicken smMLCK is conserved (Fig. 2B) and perfectly fits the canonical 6 B. Viollet, unpublished data. FIGURE 2. Identification of the smMLCK phosphorylation site for AMPK. A, chicken gizzard smMLCK was phosphorylated in vitro by AMPK. After SDS-PAGE, phosphorylated MLCK was cut from the gel and digested with trypsin. Peptides were separated by reverse-phase narrow-bore HPLC in a linear acetonitrile gradient, and fractions were counted by Č erenkov radiation. The identity of the phosphorylation site in the radiolabeled peak is indicated. B, shown is a sequence alignment of the C-terminal domain of smMLCK from chicken, human, bovine, mouse, rabbit, and sheep. Protein kinase phosphorylation sites are indicated in boldface, and numbering refers to the chicken sequence. PKC, protein kinase C; CaMKII, Ca 2ϩ /CaM-dependent kinase II.
AMPK substrate recognition motif, (X,␤)XX(S/T)XXX, where is a hydrophobic residue (Met, Val, Leu, Ile, or Phe), ␤ is a basic residue (Arg, Lys, or His), and the parentheses indicate that the order of residues at the P-4 and P-3 positions is not critical (36).
It was recently reported that in Drosophila melanogaster in which its single AMPK catalytic subunit isoform had been deleted, epithelial cell polarity was disrupted in a manner similar to the knock-out of LKB1. Moreover, AMPK was proposed to directly phosphorylate MLC at the same residue phosphorylated by smMLCK, thereby participating in the establishment of cell polarity (37). However in our hands, treatment of ASMCs with 5-amino-4-imidazolecarboxamide riboside or the A-769662 (Abbott) AMPK activator (4) led to AMPK activation, which was not associated with an increase in MLC phosphorylation (supplemental Fig. 3).
In blood vessels, metabolic stress or hypoxia can induce vasodilatation, increasing oxygen availability in peripheral tissues. Endothelium-derived relaxing factors probably play a significant role in this respect. AMPK, which is activated by hypoxia due to the rise in AMP, could mediate vasodilatation via phosphorylation-induced activation of endothelial nitric-oxide synthase, thereby increasing NO production (38). In blood vessels devoid of endothelium, AMPK activation due to metabolic A, ASMCs were preincubated with 10 M STO-609 for 60 min prior to treatment for 5 min with 10 Ϫ7 M vasopressin (Vaso) or 10 Ϫ7 M endothelin-1 (Endo-1) or for 10 min with 10 Ϫ7 M angiotensin II (AgII) and cell lysis. ASMC lysates (100 g of protein) were immunoprecipitated with anti-AMPK ␣1/␣2-subunit antibodies for AMPK assay or analyzed by immunoblotting with anti-phospho-Thr 172 AMPK ␣-subunit antibody or a mixture of anti-AMPK ␣1/␣2-subunit antibodies (anti-AMPK) as a loading control for detection with infrared-conjugated secondary antibodies and Odysseyா imaging. The results are the means Ϯ S.E. of three independent experiments. *, significant difference (p Ͻ 0.05, unpaired t test) with respect to the value corresponding to treatment without inhibitor. B, ASMCs were incubated with 10 Ϫ8 M A23187 for the indicated times prior to cell lysis. C, ASMCs were incubated with 10 Ϫ7 M vasopressin (2 min) with or without 5 mM EGTA as indicated. In B and C, AMPK activity was measured after precipitation of extracts with 10% (w/v) polyethylene glycol 6000. AMPK activity is expressed as milliunits/mg of cell lysate protein, and the values are the means Ϯ S.E. of three independent experiments. *, significant difference (p Ͻ 0.05, unpaired t test) compared with the controls. . Expression of CaMKK␤ in ASMCs, brain, and HeLa cells. Extracts from ASMCs (20 g), brain (4 g), and HeLa cells (30 g) were subjected to SDS-PAGE followed by immunoblotting with anti-CaMKK␤ antibody. The CaMKK␤1 and CaMKK␤3 isoforms are predominantly expressed in brain and HeLa cells, respectively.   . Effect of phenylephrine stimulation and KCl-induced depolarization on tension development in rat aortic rings from wild-type and ␣1-AMPK knock-out mice. Aortic rings (Ϯ2 mm) were dissected and mounted between two hooks under a tension of 20 millinewtons (mN) as described under "Experimental Procedures." Muscle tone was measured using an isometric force transducer. The artery segment was incubated for 30 min in Krebs bicarbonate solution and then stimulated to contract with phenylephrine at concentrations ranging from 10 Ϫ9 to 10 Ϫ5 M (A) or depolarized with KCl (10 -100 mM) (B). The results are the means Ϯ S.E. of five (KCl-induced depolarization) and seven (phenylephrine stimulation) individual experiments (two aortic rings/mouse). Ⅺ, ␣1-AMPK knock-out mice; f, wild-type mice. The two curves were significantly different (p Ͻ 0.05) as assessed by one-way analysis of variance. The increase in tension relative to the increase in intracellular Ca 2ϩ is given for phenylephrineinduced (C) and KCl-induced (D) contraction in aortic rings from wild-type (WT) and ␣1-AMPK knock-out (KO) mice. *, significant difference (p Ͻ 0.05, unpaired t test) with respect to the controls.