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Am J Physiol Heart Circ Physiol 293: H2911-H2918, 2007. First published August 24, 2007; doi:10.1152/ajpheart.00217.2007
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ROK contribution to endothelin-mediated contraction in aorta and mesenteric arteries following intermittent hypoxia/hypercapnia in rats

Kyan J. Allahdadi, Benjimen R. Walker, and Nancy L. Kanagy

Department of Cell Biology and Physiology, Vascular Physiology Group, University of New Mexico, Health Sciences Center, Albuquerque, New Mexico

Submitted 19 February 2007 ; accepted in final form 13 August 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We reported previously that intermittent hypoxia with CO2 to maintain eucapnia (IH-C) elevates plasma endothelin-1 (ET-1) and arterial pressure. In small mesenteric arteries (sMA; inner diameter = 150 µm), IH-C augments ET-1 constrictor sensitivity but diminishes ET-1-induced increases in intracellular Ca2+ concentration, suggesting IH-C exposure increases both ET-1 levels and ET-1-stimulated Ca2+ sensitization. Because Rho-associated kinase (ROK) can mediate Ca2+ sensitization, we hypothesized that augmented vasoconstrictor sensitivity to ET-1 in arteries from IH-C-exposed rats is dependent on ROK activation. In thoracic aortic rings, ET-1 contraction was not different between groups, but ROK inhibition (Y-27632, 3 and 10 µM) attenuated ET-1 contraction more in IH-C than in sham arteries (50 ± 11 and 78 ± 7% vs. 41 ± 12 and 48 ± 9% inhibition, respectively). Therefore, ROK appears to contribute more to ET-1 contraction in IH-C than in sham aorta. In sMA, ROK inhibitors did not affect ET-1-mediated constriction in sham arteries and only modestly inhibited it in IH-C arteries. In ionomycin-permeabilized sMA with intracellular Ca2+ concentration held at basal levels, Y-27632 did not affect ET-1-mediated constriction in either IH-C or sham sMA and ET-1 did not stimulate ROK translocation. In contrast, inhibition of myosin light-chain kinase (ML-9, 100 µM) prevented ET-1-mediated constriction in sMA from both groups. Therefore, IH-C exposure increases ET-1 vasoconstrictor sensitivity in sMA but not in aorta. Furthermore, ET-1 constriction is myosin light-chain kinase dependent and mediated by Ca2+ sensitization that is independent of ROK activation in sMA but not aorta. Thus ET-1-mediated signaling in aorta and sMA is altered by IH-C but is dependent on different second messenger systems in small vs. large arteries.

sleep apnea; endothelin-1; vascular smooth muscle cells; Rho-associated kinase

SLEEP APNEA IS A COMMON DISEASE affecting up to 20% of Americans (30) and is associated with many cardiovascular diseases including systemic and pulmonary hypertension, coronary artery disease, stroke, and cardiac arrhythmias (35). Although the exact mechanisms by which sleep apnea contributes to these disease states have yet to be defined, several studies show that sleep apnea is associated with increased levels of circulating endothelin-1 (ET-1) (18, 29, 43). Furthermore, we have demonstrated that vasoconstrictor sensitivity to ET-1 is increased in a rat model of sleep apnea (19) and that ET-1 antagonism lowers blood pressure to control levels in rats treated with intermittent hypoxia with CO2 to maintain eucapnia (IH-C). Therefore, elevated circulating ET-1 combined with increased constrictor sensitivity to the peptide may contribute to vascular pathologies in this condition.

Previously, we reported that IH-C exposure augments constrictor sensitivity to ET-1 in small mesenteric arteries (sMA) but does not increase the constrictor sensitivity to either phenylephrine (PE) or depolarizing levels of K+ (1). Additionally, we observed that the augmented constriction to ET-1 in sMA from IH-C rats is not accompanied by greater increases in intracellular Ca2+ concentration ([Ca2+]i) (1). This suggests that IH-C exposure alters the ET-1 signaling in sMA to increase receptor activation of Ca2+ sensitization. Ca2+ sensitization regulates vascular tone by increasing phosphorylation of 20-kDa myosin regulatory light chain (MLC20) independent of changes in [Ca2+]i (38). The degree of MLC20 phosphorylation is determined by both MLC kinase (MLCK) and MLC phosphatase (MLCP) activity. MLCK phosphorylates MLC20 at Ser19, initiating cross-bridge cycling between actin filaments and myosin, which results in smooth muscle contraction. MLCP removes the phosphate and thus reduces actin and myosin binding and smooth muscle contraction. Constriction can be initiated by MLCK activation after increases in [Ca2+]i (38) or by MLCP inhibition by protein kinase C (PKC), Rho-associated kinase (ROK) (39), or other Ca2+-independent kinases (16). Of these pathways, ROK has been strongly implicated in vascular smooth muscle contraction (20, 21, 34).

RhoA and ROK can be activated by many agonists, including ET-1 (25, 31, 33). RhoA, a small monomeric GTPase, becomes active after exchange of bound GDP for GTP and translocates to the plasma membrane where it stimulates ROK translocation and activation (20). Active ROK, a serine/threonine kinase, inhibits MLCP through phosphorylation of the myosin-binding regulatory subunit (2). Although ET-1 has been shown to signal through both PKC and ROK to inhibit MLCP, the present study is focused on the contribution of ROK-mediated Ca2+ sensitization because this pathway has previously been shown to be upregulated in several forms of hypertension (4, 9, 23).

To test the hypothesis that ROK contributes more to ET-1-mediated constriction in IH-C than in sham arteries, endothelium-disrupted arteries were exposed to increasing concentrations of ET-1 in the presence or absence of ROK inhibitors (Y-27632 and HA-1077). Because most studies that have examined ROK upregulation in hypertension have been conducted in conduit arteries (26, 41) and differences have been reported in the role of ROK in large vs. small arteries (3), we examined the effect of ROK inhibition in both fifth-order sMA and the larger thoracic aorta. Ionomycin-permeabilized sMA from IH-C and sham rats were used in some studies to more directly examine ET-1-stimulated Ca2+ sensitization. Expression levels and ET-1-stimulated activation of {alpha}-ROK in IH-C and sham sMA were examined using Western blots. Finally, the contribution of MLCK to ET-1-induced vasoconstriction was investigated with the use of a selective inhibitor.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male Sprague-Dawley rats (weighing 250–300 g) were used for all studies. The IH-C protocol was as described previously (1, 19). Briefly, animals were housed in Plexiglas boxes with free access to food and water and exposed to either IH-C (90-s stream of N2-CO2 to reach a nadir of 5% O2-5% CO2 followed by 90-s air flush) or sham treatment (air-air cycling), totaling 20 cycles/h for 7 h/day. A nadir of 5% O2 was chosen to simulate the systemic hypoxemia observed in patients with severe sleep apnea, where minimum O2 saturations of ~70% are seen (35). The CO2 level was chosen to maintain eucapnia (unpublished observations). Systolic blood pressure (SBP) and heart rate were recorded on days 0 and 14 to confirm our previous observation that this IH-C protocol increases blood pressure (1). SBP was recorded before the start of the daily IH-C or air-air exposure using a standard tail-cuff apparatus (IITC). Body weight was recorded to determine the effect of the exposure protocol on weight gain. Approximately 16 h after the final IH-C exposure, animals were deeply anesthetized with pentobarbital sodium (150 mg/kg), and sMA and thoracic aorta were collected for constrictor studies.

All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of New Mexico Health Science Center and conform to National Institutes of Health guidelines for animal use.

Aortic ring contraction studies. Thoracic aortas were collected from IH-C and sham rats and placed in chilled physiological saline solution (PSS) (in mmol/l: 129.8 NaCl, 5.4 KCl, 0.83 MgSO4, 19 NaHCO3, 1.8 CaCl2, 5.5 glucose, and 0.5 EGTA). Arteries were cleaned of connective tissue and surrounding fat and cut into 3-mm segments. Endothelial cells were removed by gently rubbing the lumen with fine-tipped forceps. Tissue rings were suspended in a bath containing warmed PSS maintained at 37°C and bubbled with 21% O2-6% CO2-73% N2 using metal hooks attached to Grass FT03 force transducers connected to amplifiers that fed into a data-acquisition program (Dataq, 3.01). Rings were stretched with 2,500 mg of passive tension and equilibrated for 60 min. After equilibration, viability was confirmed by contraction to PE (10–6 mol/l), and endothelial integrity was assessed by relaxation to ACh (10–6 mol/l) in rings contracted with PE. Segments relaxing <5% were used. Tissues were washed repeatedly over 60 min to remove PE and ACh and then exposed to increasing concentrations of ET-1 (10–10 to 10–7 mol/l) in the presence or absence of the ROK inhibitor Y-27632 (3 or 10 µmol/l). Contractions are expressed as %PE (10–6 M) contraction (8).

Pressurized mesenteric artery preparation. The intestinal arcade was removed and placed in a Silastic-coated Petri dish containing chilled PSS. Fifth-order artery segments were dissected from the mesenteric vascular arcade and placed in fresh PSS oxygenated with normoxic gas (21% O2-6% CO2-73% N2). Cleaned arterioles were transferred to a vessel chamber (Living Systems), cannulated with glass micropipettes, and secured with silk ligatures. The vessels were slowly pressurized to 60 mmHg with PSS using a servo-controlled peristaltic pump (Living Systems) and superfused with oxygenated 37°C PSS at 5 ml/min.

Endothelium disruption. The sMA endothelium was disabled in all experiments by passing 1 ml of air through the lumen. The integrity of the endothelium was assessed before and after disruption by exposing PE-constricted arterioles (10–6 mol/l) to ACh (10–6 mol/l). ACh-mediated vasodilation was eradicated in vessels with successfully disabled endothelium.

Vessel wall [Ca2+]i detection. After arteries were denuded, pressurized sMAs (inner diameter of sham arteries = 143.0 ± 4.1 µm; inner diameter of IH-C arteries = 143.3 ± 4.5 µm) were loaded with the cell-permeable ratiometric Ca2+-sensitive fluorescent dye fura 2-AM (Molecular Probes). Fura 2-AM was dissolved in anhydrous DMSO (1 mmol/l). Directly before loading, fura 2-AM was mixed with a 20% solution of pluronic acid in DMSO and then added to PSS, yielding a final concentration of 2 µmol/l fura 2-AM and 0.05% pluronic acid. Pressurized sMA were incubated 45 min in the dark at room temperature in air-bubbled fura 2-AM solution. After incubation, sMA were washed with 37°C PSS for 15 min to remove excess dye and to allow complete deesterification of the compound. Fura 2-loaded vessels were alternately excited at 340 and 380 nm at a frequency of 10 Hz with a dual-excitation light source (IonOptix Hyperswitch), and the respective 510-nm emissions were collected with a photomultiplier tube [ratio of 340-nm to 380-nm fluorescence (F340/F380)]. Background-subtracted emission F340/F380 results were calculated with Ion Wizard software (IonOptix) and recorded continuously throughout the experiment with simultaneous measurement of inner diameter from bright-field images as described previously (27).

Constrictor studies. After baseline internal diameter and F340/F380 were determined, constrictor studies to ET-1 (10–10 to 10–8 mol/l) were conducted in sham and IH-C sMA as reported previously (1). The role of ROK in ET-1-mediated constriction was investigated with sMA pretreated with the ROK inhibitors Y-27632 (3 µM; Calbiochem) or HA-1077 (fasudil, 10 µM; Calbiochem) at concentrations shown to be effective at preventing ROK activation (12, 32). After 10-min incubation with inhibitors, sMA were exposed to increasing concentrations of ET-1 (10–10 to 10–8 mol/l). Vessels were exposed to each concentration of agonist for 5 min in the continued presence of the inhibitors, and each artery was used for only one concentration-response curve. After ET-1 exposure, sMA were superfused with Ca2+-free PSS to cause complete relaxation and to demonstrate that the constriction was caused by reversible active tone. Constrictions are expressed as percent baseline diameter. Vessel wall Ca2+ concentration is expressed as F340/F380 because the ratio is linearly related to true molar Ca2+ concentration when the dissociation constant of fura 2 does not differ between treatment groups (14).

Permeabilized studies. Arteries were prepared as described above. After fura 2-AM loading, vessels were superfused with Ca2+-free PSS. Once vessels were completely dilated, 3 µM ionomycin (Calbiochem) was added to the Ca2+-free superfusate to permeabilize the vessel to Ca2+. After diameter and F340/F380 equilibrated, 300 nM Ca2+ was added back to the superfusate to clamp [Ca2+]i at approximate basal levels. Ca2+-clamped vessels were treated with vehicle, Y-27632 (3 µM), or the myosin light-chain kinase inhibitor ML-9 (100 µmol/l) for 10 min and then exposed to increasing ET-1.

To determine whether the entire vascular wall was penetrated by the ionomycin, some vessels were loaded intraluminally with fura 2-AM for 15 min and then washed with PSS to remove extracellular dye. The pressurized vessel was then permeabilized with ionomycin (3 µM) in the superfusate and equilibrated in PSS with a calculated free Ca2+ concentration of 300 nM as described above. The free Ca2+ concentration for all studies was calculated with the use of the Kd of EGTA for Ca2+ of 43.7 nM and the Kd of EGTA for Mg2+ of 3.33 mM at 37°C and pH 7.4 (13).

Protein analysis. To determine whether IH-C alters expression or activation of ROK in sMA, protein levels were evaluated as described previously (1). Briefly, mesenteric artery cascades (2nd- to 5th-order inclusive) from sham and IH-C rats were cleaned of connective and adipose tissue, incubated with either vehicle or ET-1 (10–8) for 30 min at 37°C, frozen in liquid nitrogen, and homogenized on ice in Tris·HCl buffer (pH 7.4) containing protease inhibitors. Homogenates were centrifuged at 1,500 g (4°C, 10 min) to remove insoluble material and then separated into cytosolic and particulate fractions by high-speed centrifugation (100,000 g at 4°C for 60 min). The cytosolic fraction was collected, and the particulate fraction was reconstituted in 100 µl of Laemmli buffer. Proteins were separated in 4–20% gradient polyacrylamide gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes. Membranes were blocked in TBS with 0.05% Tween 20 detergent (TTBS) and 1% BSA (Sigma). After blocking was completed, membranes were incubated with a mouse monoclonal antibody specific for {alpha}-ROK II (1:500; BD Biodesign) in TTBS containing 1% BSA. Membranes were incubated with peroxidase-labeled goat anti-mouse IgG (1:5,000; Bio-Rad), washed in TTBS, and then analyzed with chemiluminescence labeling (Amersham). Membranes were exposed on Kodak BIOMAX film for 3–10 min. {alpha}-ROK band densities (SigmaGel; SPSS) were normalized to the total protein loaded per lane as determined by Coomassie blue staining of the membranes.

Statistical analysis. Constriction and Ca2+ concentration-response curves were analyzed by two-way repeated-measures ANOVA with Student-Newman-Keuls post hoc test for differences between groups and treatments. The EC50 value or the concentration that produces 50% of the maximum response (Emax) was calculated for each concentration-response curve by nonlinear curve fitting using the Prism 3 GraphPad program, and group averages compared by one-way ANOVA with Student-Newman-Keuls post hoc test. Percent data were transformed to the square root of the arcsin before analysis to approximate a normal distribution (SigmaStat software; SPSS). P < 0.05 was considered statistically significant for all analyses.


   RESULTS
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 ABSTRACT
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 RESULTS
DISCUSSION
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 REFERENCES
 
Table 1 shows a significant increase in SBP in rats exposed to IH-C for 14 days. Additionally, body weight (g) increased similarly in sham and IH-C rats over the 14 days of treatment.


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  		Table 1. Baseline SBP and body weight for IH-C and sham rats

 
Mesenteric arteries from IH-C rats constrict more to ET-1 (10–10 to 10–8 mol/l) than those from sham arteries (Fig. 1A) without an increase in vessel wall Ca2+ concentration (Fig. 1B), consistent with our previous observations (1). The data in Table 2 further demonstrate that the maximum constriction (Emax) in IH-C sMA is greater than that in sham sMA, whereas the EC50 is not different.


Figure 1
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  		Fig. 1. Intermittent hypoxia with CO2 to maintain eucapnia (IH-C) exposure augments endothelin-1 (ET-1)-mediated constriction of endothelium-disrupted mesenteric arteries. Shown are simultaneous measurements of concentration-dependent constriction (A) and increases in vessel wall intracellular Ca2+ concentration ([Ca2+]) (B) to ET-1 in mesenteric arteries with disabled endothelium from sham (n = 5) and IH-C (n = 5) rats. A: constrictions are expressed as %vasoconstriction with 100% equivalent to a closed lumen. B: vessel wall intracellular [Ca2+] is expressed as the ratio of fura 2 fluorescence [ratio of 340-nm to 380-nm fluorescence (F340/F380)]. *Significant difference from sham rats.

 

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  		Table 2. EC50 and Emax for ET-1 contraction in arteries from IH-C and sham rats

 
Effects of ROK inhibition on ET-1-mediated contraction in thoracic aorta. In vehicle-treated arteries, ET-1-induced contraction was similar in sham and IH-C aortic rings. ROK inhibition with Y-27632 (3 and 10 µmol/l) diminished ET-1-mediated contraction by 41 ± 11 and 48 ± 9% in sham aorta and by 50 ± 11 and 75 ± 7% in the IH-C aorta (Fig. 2 and Table 2).


Figure 2
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  		Fig. 2. Rho-associated kinase (ROK) inhibition (Y-27632) diminishes ET-1-mediated contraction in thoracic aorta. Shown are concentration-dependent increases in isometric contraction by ET-1 in sham (A) and IH-C (B) endothelium-denuded thoracic aortic rings (3 mm) in the absence (vehicle) or presence of Y-27632 (3 or 10 µM); n = 7 experiments for all conditions.

 
Effect of ROK inhibition on ET-1-mediated vasoconstriction in sMA. In sham sMA, ROK inhibition with Y-27632 (3 µmol/l) did not diminish ET-1-mediated constriction. Instead, constriction appeared to be slightly increased (Fig. 3A). In IH-C sMA, the EC50 for the ET-1-initiated constriction was slightly but not significantly reduced in the presence of the ROK inhibitor, but constriction at several concentrations was significantly less in the presence of the inhibitor (Fig. 3B and Table 2).


Figure 3
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  		Fig. 3. ROK inhibition (Y-27632) affects ET-1-mediated constriction differently in sham and IH-C mesenteric arteries. Shown are concentration-dependent increases in constriction by ET-1 in sham (A) and IH-C (B) endothelium-disabled mesenteric arteries in the absence (vehicle) or presence of Y-27632 (3 µM). *Significant difference from vehicle (P < 0.05).

 
The ROK inhibitor HA-1077 (10 µmol/l) also appeared to slightly augment ET-1-mediated constriction in sham sMA (Fig. 4A) and diminished ET-1-mediated constriction in IH-C sMA, but again EC50 and Emax results were not significantly altered (Fig. 4B).


Figure 4
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  		Fig. 4. ROK inhibition by HA-1077 affects ET-1-mediated constriction slightly in mesenteric arteries. Shown are results for concentration-dependent constriction by ET-1 in sham (A) and IH-C (B) endothelium-disabled mesenteric arteries in the absence (vehicle) or presence of HA-1077 (10 µM). *Significant difference from vehicle (P < 0.05).

 
In addition, Ca2+ sensitivity was examined in permeabilized sMA with [Ca2+]i held constant. Arteries were permeabilized with ionomycin (3 µM), and superfusate [Ca2+]i was held constant at 300 nM to keep F340/F380 results similar to those in nonpermeabilized arteries at baseline. In permeabilized sMA, ROK inhibition did not affect ET-1-mediated constriction in either sham (Fig. 5A) or IH-C arteries (Fig. 5B). Vessel wall Ca2+ concentration ratios were unchanged throughout the protocol (Fig. 5, C and D), indicating that arteries were successfully permeabilized in the presence of 3 µM ionomycin.


Figure 5
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  		Fig. 5. ROK inhibition (Y-27632) does not diminish ET-1-mediated constriction in permeabilized mesenteric arteries. Concentration-dependent increases in constriction of sham (A) and IH-C (B) arteries and increases in vessel wall [Ca2+] of sham (C) and IH-C (D) arteries to ET-1 in cannulated, pressurized, endothelium-disabled mesenteric arteries. All arteries were permeabilized with 3 µM ionomycin and Ca2+ clamped at 300 nM in the presence of vehicle or 3 µM Y-27632 before ET-1 administration.

 
Effects of MLCK inhibition on ET-1-mediated vasoconstriction. Clamping [Ca2+]i at basal levels did not affect ET-1-mediated constriction in IH-C sMA compared with unclamped arteries. However, clamping [Ca2+]i at basal levels reduced ET-1-mediated constriction in sham arteries compared with unclamped arteries (Emax for sham arteries = 82 ± 5% and 72 ± 4% and Emax for IH-C arteries = 100 ± 7 and 99 ± 6%, intact and permeabilized, respectively; Table 2), suggesting that maximal ET-1 constriction was more dependent on [Ca2+]i and thus MLCK activation in sham sMA. To determine whether vasoconstriction by ET-1 was independent of Ca2+ activation of MLCK in the IH-C sMA, permeabilized sMA were incubated with the MLCK inhibitor ML-9 (100 µM), with superfusate [Ca2+]i at 300 nM. ML-9 dramatically reduced ET-1-mediated constriction in both sham and IH-C sMA (Fig. 6, A and B) without any change in [Ca2+]i (Fig. 6, C and D), suggesting MLCK mediates the constrictions. The effects of IH-C on vascular sensitivity to ET-1 are summarized in Table 2. There was an increase in Emax in IH-C sMA but not aorta compared with sham controls. ROK inhibition did not affect Emax in sMA; however, in aorta, ROK inhibition decreased it significantly, more in IH-C than in sham (52 ± 9 and 25 ± 7% in IH-C vs. sham). Permeabilization decreased Emax in sham but not IH-C sMA.


Figure 6
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  		Fig. 6. Myosin light chain kinase inhibition eliminates ET-1-mediated constriction in permeabilized mesenteric arteries. Shown are concentration-dependent increases in constriction (A and B) and vessel wall [Ca2+] (C and D) to ET-1 in endothelium-disabled mesenteric arteries. All arteries were permeabilized with 3 µM ionomycin and Ca2+ clamped at 300 nM in the presence of vehicle or 100 µM ML-9 before ET-1 administration. *Significant difference from vehicle (P < 0.05).

 
ROK translocation by ET-1. Finally, to determine whether ET-1 stimulates the RhoA and ROK activation in sMA, ROK levels in the cytosol and particulate fractions of homogenized sMA from IH-C and sham rats were estimated by Western analysis. In addition, some sMA were stimulated with the maximum concentration of ET-1 (10–8 mol/l) to evaluate agonist-induced translocation. Western analysis demonstrated that ROK is present in both the cytosol and particulate fractions of sham (Fig. 7A) and IH-C (Fig. 7B) sMA. However, ET-1 stimulation did not cause translocation of ROK to the particulate fraction, an observation consistent with the lack of effect of the ROK inhibitors in mesenteric artery constrictor studies (Figs. 3 and 4).


Figure 7
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  		Fig. 7. ROK is not translocated by ET-1 in mesenteric arteries. Western analysis of small mesenteric arteries from sham (A) and IH-C (B) rats (n = 5) probed for {alpha}-ROK II in cytosolic (cyto) and particulate (part) fractions. Some arteries were stimulated with ET-1 (10–8 mol/l) for 30 min before homogenization. Data are plotted as ratio of particulate to cytosolic fraction. Ratios were derived from arbitrary densitometry values, normalized to Coomassie blue staining to ensure equal loading.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
ET-1 is an endogenous peptide shown to be an important regulator of vascular smooth muscle tone, especially in pathological conditions. Our previous studies suggest that augmented ET-1 vasoconstriction contributes to IH-C-induced hypertension (1, 19), and the present study investigated a potential mechanism for this augmented response.

ET-1 initiates vasoconstriction through increases in both [Ca2+]i and Ca2+ sensitization (10, 33, 36), but our previous studies suggested that only Ca2+ sensitization is important in sMA from IH-C rats (1). The purpose of this study was to examine ET-1 signaling in vascular smooth muscle from sham and IH-C rats to determine whether increased activation of the Ca2+-sensitizing small G protein RhoA/ROK contributes to the augmented constrictor response. The main findings of this study were 1) ET-1 contraction in both sham and IH-C thoracic aortic rings is ROK dependent; 2) in sMA, ROK activation is not a major mediator of ET-1-stimulated Ca2+ sensitization in sMA, and ET-1 stimulation does not cause translocation of ROK; and 3) MLCK inhibition eliminates ET-1-mediated constriction in permeabilized IH-C and sham sMA. These data indicate that ET-1 mediates constriction through ROK in large but not in small arteries. In large arteries, the contribution of ROK appears to be increased in arteries from IH-C-treated rats but does not cause augmented vasoconstrictor sensitivity to ET-1. In contrast, ET-1 constrictor sensitivity is augmented in small arteries after IH-C treatment, but ROK appears to be a minor contributor to the response. Indeed, the effects of Y-27632 may be independent of ROK inhibition because HA-1077 (fasudil) did not have a significant effect and Y-27632 had no effect when vessel wall [Ca2+]i was maintained constant. Interestingly, we observed that in sham sMA both ROK inhibitors slightly increased ET-1-mediated constriction (Figs. 3 and 4). Although ROK in vascular smooth muscle cell has only been reported to regulate MLCP, these data suggest other signaling pathways may also be regulated by ROK, including vasodilatory pathways.

Overall, our data suggest that IH-C alters ET-1 signal transduction in both the larger aorta and smaller mesenteric arteries but has a much greater effect on vasoconstrictor sensitivity to ET-1 in small resistance arteries. This reinforces previous observations that ET-1 signaling is very different in large and small arteries (3) and is in agreement with our group's (19) previous finding that ET-1 receptor blockade lowers blood pressure in IH-C-treated rats.

In the sMA, ET-1-stimulated constriction required MLCK activation in both groups. Thus Ca2+ activation of MLCK appears universally necessary for constriction. The apparent necessity of basal levels of Ca2+ was further evidenced by the complete relaxation to Ca2+-free PSS and subsequent constriction to 300 nM Ca2+ in both groups of permeabilized sMA. However, increased [Ca2+]i appears to be required only in sham sMA to induce maximal ET-1 constriction, since holding vessel wall [Ca2+]i constant significantly diminished constriction compared with that shown in the nonpermeabilized sham arteries but not in the IH-C arteries. Together, these results suggest Ca2+ influx contributes more to constriction in sham than in IH-C sMA, consistent with our previous observations that ET-1-induced constriction in sMA from IH-C rats is independent of increases in vessel wall Ca2+ concentration (1).

Multiple studies have suggested augmented activation of ROK contributes to vascular changes in hypertension. Seko et al. (34) found that levels of active RhoA were increased in cultured aortic vascular smooth muscle cell from hypertensive compared with normotensive rats. The authors concluded that RhoA functions as a "molecular switch" in hypertension to augment constriction (34). Weber and Webb (40) similarly demonstrated a greater effect of Y-27632 to relax serotonin-induced constriction in superior mesenteric arteries from DOCA-salt hypertensive rats than in arteries from control rats. In addition, we observed that Y-27632 more effectively reverses {alpha}2-adrenoceptor-mediated constriction in aorta from chronic nitric oxide synthase inhibition hypertensive rats than in control arteries (7). Finally, the present study suggests that Y-27632 more completely prevents ET-1-dependent constriction in IH-C than in sham aortic rings. Therefore, ROK is implicated in certain vascular changes that occur in many different models of hypertension.

However, although ROK has been shown to be an important regulator of vascular tone in several different models of hypertension (15), ROK appears to play a small role in the augmented ET-1-mediated constriction of sMA from the hypertensive IH-C rats. One possibility is that alterations in ROK signaling are greater in large than in small arteries, as suggested by the present data and several earlier studies. Budzyn et al. (5) reported that ROK inhibition reduces agonist-mediated constriction in the thoracic aorta and superior mesenteric artery but only minimally affects tone in small mesenteric artery branches. Mesenteric arteries used in the present study were smaller than those in the Budzyn study (150 vs. 300 µm) and showed even less sensitivity to ROK inhibition. Therefore ET-1-induced ROK activation of Ca2+ sensitization after hypertension might be limited to large vessels, whereas other mechanisms of Ca2+ sensitization such as PKC play a more important role in small arteries (5).

Alternatively, the contribution of ROK to Ca2+ sensitization in small arteries may be agonist specific. ROK inhibition with Y-27632 significantly reduced PE-induced constriction in small femoral arteries (~130 µm) (22) and effectively reversed PE-mediated constriction but not ET-1-mediated constriction in a perfused mesenteric bed (6). Similarly, myogenic tone is ROK mediated in small cerebral arteries (17). Therefore, ROK may be activated by adrenergic agonists and myogenic tone but does not appear to be a major contributor to ET-1-induced constriction in the mesenteric resistance circulation.

The experiments with the MLCK inhibitor ML-9 were conducted to clarify whether ET-1 mediates contraction of the smooth muscle through Ca2+-dependent MLCK. Recent reports have suggested that MLC20 can be phosphorylated independent of Ca2+ and MLCK. Niiro and Ikebe (28) showed that activation of zipper-interacting protein kinase contracts rabbit mesenteric artery strips in the absence of Ca2+ and phosphorylates both Ser19 and Thr18 of MLC20 to activate cross-bridge cycling. Integrin-linked kinase similarly phosphorylates Ser19 and Thr18 to induce contraction of rat caudal artery strips (42). The dramatic decrease in ET-1-mediated constriction of mesenteric arteries with MLCK inhibition (Fig. 6) does not preclude zipper-interacting protein kinase and integrin-linked kinase participation in ET-1-mediated constriction; however, combined with the requirement for basal [Ca2+]i, it does suggest that Ca2+-activated MLCK is necessary for ET-1 constriction in these arteries.

Interestingly, oral Y-27632 lowers blood pressure to normal levels in nitro-L-arginine methyl ester-treated rats but does not affect blood pressure in control rats (34), whereas fasudil increases forearm blood flow in hypertensive patients but not in normotensive controls (24). Therefore, ROK inhibition appears to be an effective antihypertensive agent under certain conditions. However, if ROK contributes only minimally to ET-1 constriction of resistance arteries as seen here, it would not be predicted to be an effective target for treating ET-1-dependent hypertension.

In summary, the aim of the present study was to evaluate the contribution of RhoA/ROK to ET-1-mediated constriction in arteries from IH-C and sham rats. We demonstrated that ROK is not a major signaling component of ET-1-mediated vasoconstriction in IH-C mesenteric resistance arteries but contributes substantially to ET-1 contraction in the thoracic aorta. In addition, we demonstrated that Ca2+ and MLCK are necessary for ET-1-mediated constriction of both IH-C and sham sMA. Thus it appears that augmented ET-1 constrictor sensitivity in small arteries after sleep apnea-induced hypertension is MLCK dependent yet ROK independent. However, in the aorta, ET-1-mediated contraction is mediated for the most part through ROK activation.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Environmental Protection Agency Grant RD-83186001 and a Research Allocations Committee grant from the University of New Mexico. N. L. Kanagy is an Established Investigator of the American Heart Association.


   ACKNOWLEDGMENTS
 
We thank Pam Allgood and Laura Duling for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. L. Kanagy, Vascular Physiology Group, Dept. of Cell Biology and Physiology, MSC 08-4750, 1 Univ. of New Mexico, Albuquerque, New Mexico 87131 (e-mail: nkanagy{at}salud.unm.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Allahdadi KJ, Walker BR, Kanagy NL. Augmented endothelin vasoconstriction in intermittent hypoxia-induced hypertension. Hypertension 45: 705–709, 2005.[Abstract/Free Full Text]
  2. Amano M, Ito M, Kimura K, Fukata Y, Chihara K, Nakano T, Matsuura Y, Kaibuchi K. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J Biol Chem 271: 20246–20249, 1996.[Abstract/Free Full Text]
  3. Asano M, Nomura Y. Comparison of inhibitory effects of Y-27632, a Rho kinase inhibitor, in strips of small and large mesenteric arteries from spontaneously hypertensive and normotensive Wistar-Kyoto rats. Hypertens Res 26: 97–106, 2003.[CrossRef][Web of Science][Medline]
  4. Budzyn K, Marley PD, Sobey CG. Targeting Rho and Rho-kinase in the treatment of cardiovascular disease. Trends Pharmacol Sci 27: 97–104, 2006.[CrossRef][Medline]
  5. Budzyn K, Paull M, Marley PD, Sobey CG. Segmental differences in the roles of rho-kinase and protein kinase C in mediating vasoconstriction. J Pharmacol Exp Ther 317: 791–796, 2006.[Abstract/Free Full Text]
  6. Buyukafsar K, Arikan O, Ark M, Secilmis A, Un I, Singirik E. Rho-kinase expression and its contribution to the control of perfusion pressure in the isolated rat mesenteric vascular bed. Eur J Pharmacol 485: 263–268, 2004.[CrossRef][Web of Science][Medline]
  7. Carter RW, Begaye M, Kanagy NL. Acute and chronic NOS inhibition enhances {alpha}2-adrenoreceptor-stimulated RhoA and Rho kinase in rat aorta. Am J Physiol Heart Circ Physiol 283: H1361–H1369, 2002.[Abstract/Free Full Text]
  8. Carter RW, Kanagy NL. Mechanism of enhanced calcium sensitivity and {alpha}2-AR vasoreactivity in chronic NOS inhibition hypertension. Am J Physiol Heart Circ Physiol 284: H309–H316, 2003.[Abstract/Free Full Text]
  9. Chitaley K, Weber D, Webb RC. RhoA/Rho-kinase, vascular changes, and hypertension. Curr Hypertens Rep 3: 139–144, 2001.[Medline]
  10. Claing A, Shbaklo H, Plante M, Bkaily G, Orleans-Juste P. Comparison of the contractile and calcium-increasing properties of platelet-activating factor and endothelin-1 in the rat mesenteric artery and vein. Br J Pharmacol 135: 433–443, 2002.[CrossRef][Web of Science][Medline]
  11. Eto M, Kitazawa T, Yazawa M, Mukai H, Ono Y, Brautigan DL. Histamine-induced vasoconstriction involves phosphorylation of a specific inhibitor protein for myosin phosphatase by protein kinase C {alpha} and {delta} isoforms. J Biol Chem 276: 29072–29078, 2001.[Abstract/Free Full Text]
  12. Ghisdal P, Vandenberg G, Morel N. Rho-dependent kinase is involved in agonist-activated calcium entry in rat arteries. J Physiol 551: 855–867, 2003.[Abstract/Free Full Text]
  13. Goldstein DA. Calculation of the concentrations of free cations and cation-ligand complexes in solutions containing multiple divalent cations and ligands. Biophys J 235–242, 1979.
  14. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985.[Abstract/Free Full Text]
  15. Hilgers RH, Todd J Jr, Webb RC. Increased PDZ-RhoGEF/RhoA/Rho kinase signaling in small mesenteric arteries of angiotensin II-induced hypertensive rats. J Hypertens 25: 1687–1697, 2007.[CrossRef][Web of Science][Medline]
  16. Huang J, Mahavadi S, Sriwai W, Hu W, Murthy KS. Gi-coupled receptors mediate phosphorylation of CPI-17 and MLC20 via preferential activation of the PI3K/ILK pathway. Biochem J 396: 193–200, 2006.[CrossRef][Web of Science][Medline]
  17. Jarajapu YP, Knot HJ. Relative contribution of Rho kinase and protein kinase C to myogenic tone in rat cerebral arteries in hypertension. Am J Physiol Heart Circ Physiol 289: H1917–H1922, 2005.[Abstract/Free Full Text]
  18. Jordan W, Reinbacher A, Cohrs S, Grunewald RW, Mayer G, Ruther E, Rodenbeck A. Obstructive sleep apnea: plasma endothelin-1 precursor but not endothelin-1 levels are elevated and decline with nasal continuous positive airway pressure. Peptides 26: 1654–1660, 2005.[CrossRef][Web of Science][Medline]
  19. Kanagy NL, Walker BR, Nelin LD. Role of endothelin in intermittent hypoxia-induced hypertension. Hypertension 37: 511–515, 2001.[Abstract/Free Full Text]
  20. Kawano Y, Yoshimura T, Kaibuchi K. Smooth muscle contraction by small GTPase Rho. Nagoya J Med Sci 65: 1–8, 2002.[Medline]
  21. Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, Kaibuchi K. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273: 245–248, 1996.[Abstract]
  22. Koida S, Ohyanagi M, Ueda A, Mori Y, Iwasaka T. Mechanism of increased {alpha}-adrenoceptor-mediated contraction in small resistance arteries of rats with heart failure. Clin Exp Pharmacol Physiol 33: 47–52, 2006.[CrossRef][Web of Science][Medline]
  23. Lee DL, Webb RC, Jin L. Hypertension and RhoA/Rho-kinase signaling in the vasculature: highlights from the recent literature. Hypertension 44: 796–799, 2004.[Abstract/Free Full Text]
  24. Masumoto A, Hirooka Y, Shimokawa H, Hironaga K, Setoguchi S, Takeshita A. Possible involvement of Rho-kinase in the pathogenesis of hypertension in humans. Hypertension 38: 1307–1310, 2001.[Abstract/Free Full Text]
  25. Miao L, Dai Y, Zhang J. Mechanism of RhoA/Rho kinase activation in endothelin-1- induced contraction in rabbit basilar artery. Am J Physiol Heart Circ Physiol 283: H983–H989, 2002.[Abstract/Free Full Text]
  26. Moriki N, Ito M, Seko T, Kureishi Y, Okamoto R, Nakakuki T, Kongo M, Isaka N, Kaibuchi K, Nakano T. RhoA activation in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats. Hypertens Res 27: 263–270, 2004.[CrossRef][Web of Science][Medline]
  27. Naik JS, Earley S, Resta TC, Walker BR. Pressure-induced smooth muscle cell depolarization in pulmonary arteries from control and chronically hypoxic rats does not cause myogenic vasoconstriction. J Appl Physiol 98: 1119–1124, 2005.[Abstract/Free Full Text]
  28. Niiro N, Ikebe M. Zipper-interacting protein kinase induces Ca2+-free smooth muscle contraction via myosin light chain phosphorylation. J Biol Chem 276: 29567–29574, 2001.[Abstract/Free Full Text]
  29. Phillips BG, Narkiewicz K, Pesek CA, Haynes WG, Dyken ME, Somers VK. Effects of obstructive sleep apnea on endothelin-1 and blood pressure. J Hypertens 17: 61–66, 1999.[Web of Science][Medline]
  30. Phillips BG, Somers VK. Hypertension and obstructive sleep apnea. Curr Hypertens Rep 5: 380–385, 2003.[Web of Science][Medline]
  31. Sakai H, Chiba Y, Misawa M. Role of Rho kinase in endothelin-1-induced phosphorylation of CPI-17 in rat bronchial smooth muscle. Pulm Pharmacol Ther. In press.
  32. Sakamoto K, Hori M, Izumi M, Oka T, Kohama K, Ozaki H, Karaki H. Inhibition of high K+-induced contraction by the ROCKs inhibitor Y-27632 in vascular smooth muscle: possible involvement of ROCKs in a signal transduction pathway. J Pharm Sci 92: 56–69, 2003.[CrossRef][Web of Science]
  33. Scherer EQ, Herzog M, Wangemann P. Endothelin-1-induced vasospasms of spiral modiolar artery are mediated by rho-kinase-induced Ca2+ sensitization of contractile apparatus and reversed by calcitonin gene-related Peptide. Stroke 33: 2965–2971, 2002.[Abstract/Free Full Text]
  34. Seko T, Ito M, Kureishi Y, Okamoto R, Moriki N, Onishi K, Isaka N, Hartshorne DJ, Nakano T. Activation of RhoA and inhibition of myosin phosphatase as important components in hypertension in vascular smooth muscle. Circ Res 92: 411–418, 2003.[Abstract/Free Full Text]
  35. Shamsuzzaman AS, Gersh BJ, Somers VK. Obstructive sleep apnea: implications for cardiac and vascular disease. JAMA 290: 1906–1914, 2003.[Abstract/Free Full Text]
  36. Sirous ZN, Fleming JB, Khalil RA. Endothelin-1 enhances eicosanoids-induced coronary smooth muscle contraction by activating specific protein kinase C isoforms. Hypertension 37: 497–504, 2001.[Abstract/Free Full Text]
  37. Somlyo AP, Somlyo AV. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev 83: 1325–1358, 2003.[Abstract/Free Full Text]
  38. Somlyo AP, Somlyo AV. Signal transduction through the RhoA/ Rho-kinase pathway in smooth muscle. J Muscle Res Cell Motil 25: 613–615, 2004.[Web of Science][Medline]
  39. Weber DS, Webb RC. Enhanced relaxation to the rho-kinase inhibitor Y-27632 in mesenteric arteries from mineralocorticoid hypertensive rats. Pharmacology 63: 129–133, 2001.[CrossRef][Web of Science][Medline]
  40. Wehrwein EA, Northcott CA, Loberg RD, Watts SW. Rho/Rho kinase and phosphoinositide 3-kinase are parallel pathways in the development of spontaneous arterial tone in deoxycorticosterone acetate-salt hypertension. J Pharmacol Exp Ther 309: 1011–1019, 2004.[Abstract/Free Full Text]
  41. Wilson DP, Sutherland C, Borman MA, Deng JT, Macdonald JA, Walsh MP. Integrin-linked kinase is responsible for Ca2+-independent myosin diphosphorylation and contraction of vascular smooth muscle. Biochem J 392: 641–648, 2005.[CrossRef][Web of Science][Medline]
  42. Zamarron-Sanz C, Ricoy-Galbaldon J, Gude-Sampedro F, Riveiro-Riveiro A. Plasma levels of vascular endothelial markers in obstructive sleep apnea. Arch Med Res 37: 552–555, 2006.[CrossRef][Web of Science][Medline]



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