All studies were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University and conform to the Public Health Service Policy on Humane Care and Use of Laboratory Animals (2015) and the National Research Council “Guide for the Care and Use of Laboratory Animals” (Eighth Edition). Specific-pathogen free, male, New Zealand White rabbits (weight range: 2.8–3.8 kg, mean weight: 3.35 ± 0.23 kg, age range: 12–15 weeks) were obtained from Robinson Services, Inc., and maintained in the vivarium at 19-22°C and a 12 h light, 12 h dark cycle for at least 6–7 days prior to experimentation. Animals were individually housed, provided environmental enrichment and fed a combination of pelleted high-fiber rabbit food (Harlan Teklad 2031, ∼1 cup/day) and hay. Specific-pathogen free mice were obtained from Jackson Laboratories and maintained in the vivarium at 22–23°C and a 12 h light, 12 h dark cycle for at least 3–4 days prior to experimentation. Mice (average weights ∼23–30 g) were normally group housed (except for aggressive male mice, which were individually housed with added enrichment) and fed Envigo Teklad 7012 Rodent Diet ad lib.

Large (FA) and small (superficial inferior EA) muscular arteries perfusing musculocutaneous vascular beds, and large (RA) and small (third order MA) muscular arteries perfusing the viscera, were isolated from rabbits and prepared as described previously with minor modifications (Ratz, 1990). Also isolated were the rabbit carotid artery (CA), rabbit liver, mouse MA, mouse ileum and mouse extensor digitorum longus. Arteries were cleaned of adhering tissues by microdissection (Olympus SZX12) and cut into rings ∼2.5 mm wide. The liver was perfused with cold (∼4°C) saline prior to removal to clear the vascular bed of blood and quickly cool cells, and sliced into ∼0.5 mm thin sheets. Tissues were placed in a normal physiological salt solution (NPSS), the composition of which was, in mM, 140 NaCl, 4.7 KCl, 1.2 Na₂HPO₄-7H₂O, 2.0 MOPS, 0.02 Na₂ethylenediamine tetraacetic acid to chelate heavy metals, 5.6 D-glucose, 1.6 CaCl₂ and 1.2 MgCl₂, made with high-purity (17 MΩ) deionized water and adjusted using NaOH to a pH of 7.4 at 37°C. Each artery ring was either not stretched (retained at zero external force, F_ze) or secured in a tissue myograph (Model 610M, Danish Myo Technology) and adjusted to the optimum length for muscle contraction (L₀) using an abbreviated length-tension protocol in which tissues were contracted by addition of a physiological salt solution adjusted so that the KCl concentration would rapidly produce a maximum contraction (KPSS; 110 μM KCl substituted isosmotically in the NPSS for NaCl) (Ratz and Murphy, 1987; Ratz, 1993). For each tissue, the maximum value of the KPSS-induced contraction at L₀ was recorded as the F₀ value, and all subsequent contractile responses were normalized to this value. Thus, the strength of a subsequent response to a contractile stimulus was reported as the ratio F/F₀, where F₀ was the KPSS induced response made equal to the value “1.” Unless otherwise indicated, all experiments were conducted on denuded arteries in the presence of 100 μM L-NMMA and 30 μM ODQ to block vasodilatation due to nitric oxide release and activation of guanylyl cyclase.

Artery rings at L₀ were contracted by the step-wise, cumulative addition of increasing concentrations of one of the following five contractile agonists: Ang II, KCl (as KPSS), PE, U-46619 or VP. Rabbit arteries were used for all CRC analyses except for the effect of A-769662 (Tocri, Bio-Techne Corp., Minneapolis, MN, United States) on U-46619. Rabbit MA does not respond to Ang II or VP, and responds poorly to U-46619 (Ratz, 2016), whereas mouse MA responds strongly to U-46619. Thus, when considering MA, no data (Figures 3D,T, n.d.) was obtained for Ang II and VP CRCs, and mouse MA was used to assess the effect of A-769662 on a U-46619 CRC (Figure 3P). Permeabilized tissues were activated by addition of Ca²⁺ and GTPγS as described previously (Clelland et al., 2011). Data were reported as active force normalized to F₀ (F/F₀), and thus, an F/F₀ value of 1 was equal to the maximum KPSS-induced contraction achieved during the initial determination of L₀.

Rabbit MA was chemically permeabilized with β-escin as described previously (Clelland et al., 2011) with minor modifications. In short, each artery ring at L₀ was incubated at 30°C in a Ca²⁺-free “relaxing solution” (in mM: 74.1 potassium methanesulfonate, 4.0 magnesium methanesulfonate, 4.0 Na2ATP, 4.0 EGTA, 5.0 creatine phosphate, 30.0 PIPES, adjusted to pH 7.1 with 1 N KOH and ionic strength 180 with additional 0.5 M potassium methanesulfonate). Permeabilization was achieved by incubating tissues in β-escin (Sigma–Aldrich, St. Louis, MO, United States) dissolved in Ca²⁺-free relaxing solution at room temperature. To identify the optimum permeabilization time, tissues were exposed to 100 μM β-escin for 15, 30, 45 and 60 min, then subjected to a Ca²⁺ CRC (Figure 1A) and, after washout in a Ca²⁺-free relaxing solution, exposed to KPSS (Figure 1B). The relative responsiveness to Ca²⁺ compared to KPSS is an index of the fraction of cells effectively permeabilized. Exposure for 60 min was required to produce a strong and potent Ca²⁺ CRC response with a negligible residual response to KPSS (not different than zero, P < 0.05, Figure 1). Thus, subsequent tissues were permeabilized with 100 μM β-escin for 60 min. Following permeabilization, each tissue was washed thoroughly with Ca²⁺-free relaxing solution to remove β-escin. The calcium ionophore A23187 (10 μM) was added to deplete sarcoplasmic reticulum Ca²⁺, and 1 unit/ml of calmodulin (Sigma–Aldrich, St. Louis, MO, United States) was added to compensate for loss during permeabilization. All permeabilized tissues and solutions were maintained at room temperature.

The ability of A-769662 to activate AMPK, and to alter phosphorylation of the downstream effectors ACC, MLC, rhoA, cofilin, myosin phosphatase regulatory subunit (MYPT1), and phospholamban (PLB), was assessed by standard 1-dimensional sodium dodecylsulfate gel electrophoresis followed by “Western” blotting, enhanced chemiluminescence (Pierce, Thermo Fisher Scientific Inc., Rockford, IL, United States) and image analysis of exposed x-ray film (CLINICAELECT Blue, Carestream Health, Inc., Rochester, NY, United States) using an imager (Konica Minolta Medical & Graphic, Inc.) and image analysis software (Image J¹), as described previously (Ratz, 2001) with minor modifications. The degree of change in phosphorylation was normalized to the basal (control) phosphorylation level, and the data were presented as fold-control, unless otherwise indicated. At the appropriate time point, tissues were rapidly frozen in dry ice-cooled acetone containing 6% trichloroacetic acid, 10 μM dithiothreitol and 30 mM NaF, slowly thawed, dried, weighed and homogenized with a buffer containing 25 mM Tris- Base, 20 mM dithiothreitol, 10% glycerol, 1% sodium dodecylsulfate, 5 mM EGTA, 1 mM EDTA, 50 mM NaF, 1 mM activated Na⁺ orthovanadate, 10 μg/ml leupeptin, 10 μg/ml aprotonin and 1 mM AEBSF. Protein samples (15–20 μg/μl) resolved by gel electrophoresis and Western-blotted onto polyvinylidene difluoride membranes were probed using anti-phospho AMPK (pT172) antibody (1:1000, Cell Signaling Technology, United States), anti-phospho ACC (pS79) antibody (1:1000, Cell Signaling Technology, United States), anti-phospho cofilin (pS3) antibody (1:500, Cell Signaling Technology, United States), anti-phospho MLC (pS19) antibody (1:1000, Sigma–Aldrich, United States), anti-phospho MYPT1 (T853) antibody (1:1000, EMD Millipore, United States), anti-phospho PLB (pS16) antibody (1:1000, Badrilla, United Kingdom), and anti-phospho rhoA (pS188) antibody (1:500, Santa Cruz Biotechnology, United States) at 4°C overnight. Goat-α-rabbit IgG- horseradish peroxidase was used as secondary antibody (1:2000, Santa Cruz Biotechnology, United States) at room temperature for 1 h. All lanes were loaded with identical quantities of protein. However, two proteins expressed by housekeeping genes, β-actin and GAPDH, were routinely monitored to assess uniform protein loading across lanes. Data that did not display the expected equivalent protein loading were discarded. For these proteins, blots were incubated with β-actin antibody (C4) horseradish peroxidase (1:100000, Santa Cruz, CA, United States) and GADPH antibody (1:5000, Santa Cruz, CA, United States) at room temperature for 1 h.

In some experiments, the natural compound BBR, anti-atherogenic drug SIM, and anti-diabetic drug MET were used for comparison with A-769662 because these agents are reported also to activate AMPK (Ewart and Kennedy, 2011; Hardie, 2013). In the chemical permeabilization experiment, the Ca²⁺-calmodulin-MLC kinase inhibitor trifluoperazine (TFP), and ROCK inhibitor HA-1077, were used for comparison with A-769662. Protein kinase A (PKA) and protein kinase G (PKG) activation are well-known to cause VSM relaxation. Thus, for a comparison, forskolin (FSK) was used to activate adenylyl cyclase and, thus, cAMP and PKA, and 8-bromo-cGMP (8b-cGMP) was used to activate PKG. Cyclopiazonic acid (CPA) was used to inhibit the sarcoplasmic and endoplasmic reticulum Ca²⁺-ATPase (SERCA), and 3 μM YM-58483 (a.k.a., BTP-2), was used to block store-operated Ca²⁺ channels (Harper and Poole, 2011) because this drug displays marked selectivity for inhibition of store-operated over non-selective Ca²⁺ channels. Moreover, YM-58483 selectively inhibits TRPC channels over TRPV channels, two proteins implicated in forming store-operated Ca²⁺ channels (He et al., 2005). All data were analyzed using Graph Pad Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, United States) and are presented as mean ± standard error of the mean (SE). CRC data for each tissue and each contractile agonist were fitted to a sigmoidal curve (Kenakin, 1997), except for tissues strongly inhibited by SIM, where force values at each agonist concentration were compared. For statistical analyses, and as indicated in the figure legends, data (“test” and “control”) were evaluated by the One-Sample t-test, Student’s t-test or Analysis of Variance (ANOVA) and the Dunnett’s post hoc test, and the Null hypothesis was rejected at P < 0.05. When applying the t-test two times, the Bonferroni correction was applied.

A-769662 binds the β1 subunit of AMPK, and there is evidence that A-769662 may act only on AMPK heterotrimers comprised of α₂β₁ subunits (Timmermans et al., 2014). Thus, to determine whether AMPK α₂ and especially AMPK β₁ subunits are expressed in arteries, the relative expression of these subunits was examined by comparing α₁/α₂ and β₁/β₂ expression ratios in muscular arteries (FA, EA, RA, and MA) and the large elastic CA using Western blot analysis. Expression of β₁ was equal to expression of β₂ in all arteries except FA, in which β₁ expression was slightly but significantly less than β₂ (Figure 2A). The expression of α₂ was ∼2-fold that of α₁ in all arteries except rabbit MA, which expressed approximately equal levels of α₁ and α₂ subunits (Figure 2B). The low α₁/α₂ expression of arterial muscle was not due to the inability of α₁ antibody to detect α₁ because, using the same antibodies and ratiometric analysis, we found that mouse ileum expressed nearly 100% α₁, and conversely, that mouse extensor digitorum longus (EDL) expressed nearly 100% α₂ (Figure 2C). Based on these data, there was no a priori reason to suspect that A-769662 would not be capable of inhibiting contractions induced in the rabbit arteries examined.

Once activated, AMPK is proposed to cause relaxation (inhibition of contraction) of large elastic arteries (e.g., aorta) and not smaller muscular arteries by an endothelium-independent mechanism (Goirand et al., 2007; Davis et al., 2012). However, A-769662 was shown to inhibit small muscular arteries of the mouse and hamster (Schneider et al., 2015). Thus, the ability of A-769662 to inhibit both large and small muscular arteries was examined to determine whether A-769662 can effectively relax muscular arteries known to display functional differences. In particular, this experiment analyzed the ability of A-769662 to inhibit contractions induced by Ang II (Figures 3A–C), KCl (Figures 3E–H), PE (Figures 3I–L), U-46619 (Figures 3M–P), and VP (Figures 3Q–S) in several different muscular arteries, including large musculocutaneous (FA, Figure 3, first column) and visceral (RA, Figure 3, third column) arteries, and small musculocutaneous (EA, Figure 3, second column) and visceral (MA, Figure 3, fourth column) arteries. In both large and small musculocutaneous arteries, A-769662 strongly inhibited contractions induced by Ang II and U-46619, and modestly but significantly inhibited contractions induced by VP. By contrast, the weak VP contractile response induced in the visceral RA was not inhibited by A-769662. Also by contrast, A-769662 had no effect on contractions induced by PE in musculocutaneous arteries, but did cause a reduction in the contractile potency of visceral arteries. In visceral arteries, 30 μM A-769662 did not reduce the maximum efficacy of contraction. This was true even when employing higher A-769662 concentrations (100 and 300 μM, Figures 3K,L). Moreover, A-769662 only weakly inhibited KCl-induced contractions in the larger FA and RA, and had no effect on KCl-induced contractions in the smaller EA and MA.

In summary, A-769662 induced a broad range of relaxant effects, from very strong inhibition to no inhibition of muscular arteries, and in nearly every case where A-769662 exerted inhibition of contraction, higher concentrations of stimulus overcame the inhibition. In short, A-769662 displayed differential inhibitory activity when considering (1) stimulus-type, (2) musculocutaneous vs. visceral arteries, and (3) large vs. small muscular arteries. Together, these data do not support a mechanism of A-769662-induced inhibition of contraction that involves a signaling mechanism downstream from and common to all of the contractile stimuli and all of the vascular segments examined, such as MLCK and ROCK.

To determine whether 30 μM A-769662 could cause an NO-cGMP-mediated relaxation in EA, tissues not denuded of endothelium and not treated with ODQ nor L-NMMA were contracted in a step-wise fashion with increasing PE concentrations. CRCs produced in the presence of A-769662 were not different than control data, indicating that A-769662 did not cause relaxation of a PE-induced contraction by a nitric oxide-cGMP-dependent pathway in EA (n = 3, data not shown).

Our data show that A-769662 caused strong relaxation of certain arteries. To determine whether arterial relaxation induced by A-769662 can be linked to an increase in AMPK-pT172, EA, FA, MA and RA were treated for 60 min with A-769662, and the level of AMPK-pT172 was measured and compared to control, untreated, tissues. A-769662 did not cause an increase AMPK-pT172 in any artery examined (Figure 4A). By contrast, 30 μM A-769662 induced a ∼2-fold increase in AMPK-pT172 in liver slices (Figure 4A). Two other known AMPK activators, MET and BBR, increased AMPK-pT172 above the basal level in both EA and liver, and a third known AMPK activator, SIM, increased AMPK-pT172 in liver but not in EA (Figure 4A). Although 30 and 100 μM A-769662 did not increase AMPK-pT172 in RA, starvation (no glucose added to the buffer and O₂ depletion by vigorous bubbling with 100% N₂) (Smith et al., 2017) caused a 2.2 ± 0.4-fold increase (Figure 4B, n = 3). Notably, both A-769662 and starvation caused a ∼2-fold increase in the downstream AMPK substrate ACC-pS79 in RA (Figure 4C), suggesting that A-769662 may activate AMPK without elevating the degree of AMPK-pT172 in rabbit artery.

To compare the relaxant effects of A-769662 with MET and SIM, EA was contracted in the presence and absence of 30 μM MET (Figure 5, first column) and 30 μM SIM (Figure 5, second column). A-769662, MET and SIM displayed differential effects on contraction of the small musculoskeletal muscular artery EA, ranging from no inhibition to strong inhibition [compare Figure 3 column 2 (EA) with Figure 5]. Notably, MET, which caused an increase in AMPK-pT172 in EA (see Figure 4A), had no effect on contractions induced by any stimulus examined (Figure 5, first column), and SIM that did not elevate AMPK-pT172 (see Figure 4A) caused inhibition of contractions induced by all of the stimuli tested (Figure 5, second column).

Chemical permeabilization abolishes plasma membrane Ca²⁺ regulation and permits access to the cytosol so that the cytosolic Ca²⁺ level can be directly controlled. To determine whether A-769662 can cause relaxation independently of an effect on plasma membrane Ca²⁺ regulation, MA was permeabilized with β-escin and a Ca²⁺ CRC was generated in the presence and absence of 100 μM A-769662. Whereas 100 μM A-769662 reduced the potency of a PE CRC in intact MA (Figure 6A), in β-escin-permeabilized MA, 100 μM A-769662 increased the potency and maximum efficacy of a Ca²⁺ CRC (Figures 6B, 7A). For comparison, BBR and SIM were also examined. In intact MA, 30 μM BBR, like 100 μM A-769662, reduced the potency of a PE CRC, and 30 μM SIM dramatically reduced the maximum efficacy (Figure 6A). However, neither BBR nor SIM had an effect on a Ca²⁺ CRC in permeabilized MA (Figure 6B). Together, these data indicate that when the cytosolic Ca²⁺ level is “clamped” at discrete levels (Ca²⁺-CRC in permeabilized muscle), A-769662, BBR and SIM did not cause VSM relaxation, suggesting that the relaxation of non-permeabilized (plasma membrane-intact) muscle by these agents was due to an alteration in Ca²⁺ regulation at the plasma membrane and not to signaling pathways down-stream from Ca²⁺, such as MLCK, or independent of Ca²⁺, such as ROCK.

As a positive control, the calmodulin and thus MLCK blocker TFP strongly inhibited Ca²⁺-induced contractions in permeabilized MA as expected (Figures 7Aa,Bc,Ca). To further test the hypothesis that the A-769662-induced relaxation of contraction was not due to an inhibition of signaling systems downstream from, or independent of, Ca²⁺, permeabilized MA was contracted submaximally with Ca²⁺ at pCa 6.25, then contracted further by addition of GPTγS (“G” in Figures 7Bd–f) to cause Ca²⁺ sensitization by activation of ROCK. A-769662 had no effect on the contraction induced by Ca²⁺ (Figures 7Bb,Ca) compared to control (Figure 7Ba) or that induced by GTPγS (Figure 7Be, “G”, Figure 7Cb) compared to control (Figure 7Bd). As expected, TFP strongly inhibited the Ca²⁺-induced contraction (Figures 7Bc,Ca), and the ROCK inhibitor H-1077 depressed the contraction induced by GTPγS (Figure 7Bf, “G”, Figure 7Cb). Moreover, A-769662 had no effect on the basal phosphorylation levels of MLC (Figure 8A), rhoA (Figure 8C), cofilin (Figure 8D), and MYPT1 (Figures 8E,F), and did not reduce the degree of active rhoA (Figure 8B) in FA. Likewise, A-769662 did not cause an increase in PLB-pS16 in FA as did FSK, a positive control (Figure 8G). Together, these data support the previous experiments suggesting that A-769662 did not cause inhibition of contraction by inhibiting MLCK or ROCK.

Results from chemically permeabilized artery support the hypothesis that the A-769662-induced relaxation of plasma membrane-intact artery was due to an effect on plasma membrane Ca²⁺ regulation. To test this hypothesis, the effect of A-769662 on cytosolic free Ca²⁺ was examined in fura-2-loaded artery. Concomitant with its ability to inhibit contraction (Figure 9A), 30 μM A-769662 inhibited the ability of a submaximum concentration of PE to induce a strong increase in [Ca²⁺]_i (Figure 9B). This was true both for RA (Figures 9A–D) and MA (Figures 9E,F). For a comparison, SIM also caused a strong inhibition of PE-induced contraction and increase in [Ca²⁺]_i, whereas MET did not (Figures 9C–F). Notably, A-769662 and SIM, and not MET, produced an immediate reduction in the basal level of [Ca²⁺]_i (Figure 10, and see Figure 9B). The strong reduction in force during a second contraction (Figure 9E, DMSO) that did not correlate with a reduction in [Ca²⁺]_i (Figure 9F, DMSO) in MA was likely due to receptor-activation induced desensitization (arterial memory) (Ratz, 1995, 1999).

Data from the fura-2-loaded artery experiments suggest that A-769662 caused relaxation by inhibiting Ca²⁺ mobilization. To determine whether inhibition of store-operated Ca²⁺ entry played a role in A-769662-induced relaxation, the ability of A-769662 to relax a CPA-induced contraction in FA was assessed. Inhibition of the SERCA pump by CPA causes the release of sarcoplasmic reticulum Ca²⁺ stores which, in turn, activates Ca²⁺ entry through store-operated Ca²⁺ channels located in the plasma membrane. In FA, 10 μM CPA caused an initial strong contraction that declined to a weaker steady-state response (Figures 11A,C, Control). A-769662 added during the tonic phase of a CPA-induced contraction (∼30 after CPA addition) caused an immediate relaxation (Figure 11A, 30 μM A-769), reducing the steady-state response at 90 min from ∼25% of the peak contraction to ∼10% (Figure 11B). By comparison, the selective store-operated Ca²⁺ channel inhibitor YM-58483 (a.k.a, BTP2) (Harper and Poole, 2011) also caused an immediate relaxation (Figure 11C, 3 μM YM-584) and reduced the steady-state response to ∼-10% (Figure 11D). That is, YM-58483 reduced force to a value less than the resting value, indicating that FA displayed a small degree of active tone. Together, these data suggest that A-769662 caused relaxation, in part, by immediate inhibition of store-operated Ca²⁺ channels.