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Am J Physiol Heart Circ Physiol 283: H1361-H1369, 2002. First published May 30, 2002; doi:10.1152/ajpheart.01101.2001
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Vol. 283, Issue 4, H1361-H1369, October 2002

Acute and chronic NOS inhibition enhances alpha 2- adrenoreceptor-stimulated RhoA and Rho kinase in rat aorta

Rebecca W. Carter, McKenzie Begaye, and Nancy L. Kanagy

Cell Biology and Physiology Department, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131-5218


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

We demonstrated that arteries from rats made hypertensive with chronic nitric oxide (NO) synthase (NOS) inhibition (Nomega -nitro-L-arginine in drinking water, LHR) have enhanced contractile sensitivity to alpha 2-adrenergic receptors (alpha 2-AR) agonist UK-14304 compared with arteries from normotensive rats (NR). NO may regulate vascular tone in part through suppression of RhoA and Rho kinase (ROK). We hypothesized that enhanced RhoA and ROK activity augments alpha 2-AR contraction in LHR aortic rings. Y-27632 eliminated UK-14304 contraction in LHR and NR aortic rings. The order of increasing sensitivity to Y-27632 was the following: endothelium-intact NR, LHR, and endothelium-denuded NR. UK-14304 stimulated RhoA translocation to the membrane fraction in LHR and denuded NR but not in intact NR aorta. Basally, more RhoA was present in the membrane fraction in denuded NR than in intact NR or LHR aorta. Relaxation to S-nitroso-N-acetyl-penicillamine and Y-27632 in denuded ionomycin-permeabilized rings was greater in NR than in LHR. Together these studies indicate alpha 2-AR contraction depends on ROK activity more in NR than LHR aorta. Additionally, endogenous NO may regulate RhoA activation, whereas chronic NOS inhibition appears to cause RhoA desensitization.

alpha 2-adrenergic receptors; vascular smooth muscle


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

VASCULAR SMOOTH MUSCLE contraction is dependent on increases in intracellular calcium and calcium sensitivity. Although early studies focused on the role of intracellular calcium to regulate contraction, recent emphasis has been placed on the mechanism of increasing contractile filament calcium sensitivity. Agonists activate several kinases that augment calcium sensitivity, including ERK1/2 (2, 7), protein kinase C (6, 18), and the more recently described Rho-associated kinase (ROK) (13, 32). ROK phosphorylates and deactivates myosin light chain (MLC) phosphatase, resulting in accumulation of phosphorylated MLC at constant intracellular calcium concentrations (32, 38). Rho and its target ROK participate in contraction to phenylephrine, endothelin, angiotensin, and norepinephrine (27, 28, 35, 40, 41). The study of Rho and subsequently ROK has revealed a new paradigm for the regulation of vascular smooth muscle contraction. ROK has also been shown to participate in hypertension. Initial studies demonstrated that ROK inhibitor Y-27632 given intravenously to DOCA-salt, renal-hypertensive, and spontaneously hypertensive rats decreases blood pressure more than in normotensive controls (40). Given these data, it is tempting to speculate that Rho and ROK augment calcium sensitivity in hypertension.

We previously demonstrated that endothelium-denuded arteries from chronic NOS-inhibited (with Nomega -nitro-L-arginine, L-NNA) hypertensive rats (LHR) were more reactive to KCl and to the alpha 2-adrenergic receptor (alpha 2-AR) agonist UK-14304 than were arteries from control, normotensive rats (NR) (20).

The alpha 2-AR, found in both endothelium and vascular smooth muscle (3, 34), is a G protein-coupled receptor, apparently acting through Gi (34). When bound to ligand, endothelial receptors activate endothelial nitric oxide (NO) synthase (NOS) and release NO (3, 33). However, the vascular smooth muscle receptor produces vasoconstriction (19, 18, 26). Therefore, in healthy arteries, the endothelium and smooth muscle alpha 2-AR work in concert with alpha 1-AR to produce an appropriate response to catecholamines. In chronic NOS inhibition hypertension, the increased contractile response to alpha 2-AR agonists may be mediated by augmented alpha 2-AR activation of the RhoA-ROK pathway. However, it has not been demonstrated that alpha 2-AR agonists activate Rho in vascular smooth muscle or if there is increased ROK activity with NOS inhibition hypertension. Indeed, the signaling pathway for vascular alpha 2-AR contraction is poorly defined, although some evidence suggests the alpha 2-AR contractile pathway may contain RhoA. In preadipocytes, alpha 2-AR stimulation by UK-14304 increased stress fiber formation. This response was blocked by the RhoA inhibitor C3 exoenzyme of Clostridium botulinum (4). Similarly, carbachol-stimulated actin reorganization in airway smooth muscle was blocked by C3, linking Gi protein-coupled receptors to RhoA activation (39).

Recently, it was discovered that NO donors can relax vascular smooth muscle independent of changes in intracellular calcium, suggesting that NO regulates calcium sensitivity (5, 36). Attenuation of calcium sensitivity by NO may be mediated by cGMP-dependent kinase (PKG). PKG phosphorylates RhoA in vitro and 8-bromo-cGMP, C3, and Y-27632 attenuate GTPgamma s-induced increases in calcium sensitivity in permeabilized smooth muscle (37). Additionally, recent evidence suggests NO acts as a vasodilator in part by PKG inhibition of the RhoA-ROK-MLC phosphatase pathway (17). Chronic NOS inhibition hypertension may provide some insight into the role of NO modulation of RhoA and ROK in hypertension.

Together, these data led us to hypothesize that enhanced alpha 2-AR vasoreactivity with chronic NOS inhibition hypertension results from increased RhoA and ROK activity. To test this hypothesis, we used contractile studies and Western blots with UK-14304 and Y-27632, a ROK inhibitor, in LHR and NR aortic rings. Because NO may acutely regulate RhoA and ROK activity, the effect of acute NOS inhibition was compared with changes associated with chronic NOS inhibition hypertension. These experiments look at the contribution of RhoA and ROK to alpha 2-AR contraction in NR and LHR aorta. Additionally, these studies add to the knowledge of endogenous NO regulation of RhoA.


    METHODS AND MATERIALS
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

Animals. Male Sprague-Dawley rats (250-300 g) were given tap water containing 0.5 g/l L-NNA (LHR) or vehicle (NR) to drink over 14 days. Blood pressures (tail cuff, IITC; Woodland Hills, CA) and animal weights were measured on days 0, 7, and 14. Mean systolic blood pressures on day 14 were 136 ± 3 and 201 ± 2 mmHg for NR and LHR, respectively. After the 14-day treatment period, animals were anesthetized with intraperitoneal pentobarbital sodium. Thoracic aorta were removed and placed in ice-cold physiological saline solution (PSS in mM: 130 NaCl, 4.7 KCl, 1.18 KH2PO4, 1.17 MgSO4 · 7H2O, 14.9 NaHCO3, 5.5 dextrose, 0.026 CaNa2-EDTA, and 1.6 CaCl2; pH 7.3). Vessels (mean diameter, 1.5 mm for both NR and LHR) were cleaned of visible fat and cut into 4-mm rings.

Treatments. To evaluate changes in RhoA and ROK contribution to alpha 2-AR contraction following chronic NOS inhibition hypertension, each LHR and NR aorta was divided into four rings. LHR aorta were compared with NR aorta with and without acute NOS inhibition to evaluate the effect of NOS inhibition on RhoA and ROK activity. NOS was inhibited acutely by two methods: endothelium denuding and pretreatment with 100 µM L-NNA, a concentration shown to inhibit cGMP formation by NO (15, 16). Two rings were denuded of endothelium with the closed tip of sharp forceps. The remaining two rings were left endothelium intact. One endothelium-denuded ring and endothelium-intact ring were pretreated 30 min with L-NNA (100 µM). The other endothelium-intact and endothelium-denuded rings were pretreated with vehicle (distilled, deionized H2O). All experiments, except when specified, were performed using these treatments on both NR and LHR aorta. Endothelium-intact and -denuded LHR aorta were treated similarly to NR to control for other endothelial factors and potential nonspecific effects of L-NNA. LHR endothelium-denuded and endothelium-intact data were combined when results were not different.

Contractile studies. Rings were attached to a force transducer (Grass Instruments; Quincy, MA) with stainless steel hooks, and generated tension was recorded on a polygraph (Gould Instruments; Harvard, MA). Two rings, one NR and one LHR, were placed in a water-jacketed tissue bath containing 37°C PSS bubbled with 95% O2-5% CO2. Rings were stretched to 2,500 mg passive tension and allowed to equilibrate 60 min. Indomethacin (1 µM) was added in the final 30 min. To determine viability, rings were exposed to a single concentration of phenylephrine (0.1 µM). Relaxation to acetylcholine (1 µM) in phenylephrine-contracted rings was used to check for endothelium. Phenylephrine-contracted intact LHR rings did not relax significantly to acetylcholine during tests for viability, suggesting that NOS is blocked in the hypertensive group. Mean acetylcholine relaxation in intact NR was 76%, whereas relaxation in intact LHR was 4%. Rings were washed until no active tension remained (30-60 min) and then were exposed to cumulative concentrations of the alpha 2-AR agonist UK-14304 in the presence or absence of increasing concentrations of the ROK inhibitor Y-27632. Time controls were performed with each experiment, and data were not reported if differences were apparent in time controls.

In separate experiments, viable rings were preconstricted using cumulative concentrations of UK-14304. After final contraction plateau (10 µM UK-14304), rings were exposed to cumulative concentrations of Y-27632, and maximal relaxation was measured. In time controls performed with each experiment, UK-14304 contraction was sustained or even slightly increased over the experimental period. A few rings relaxed below baseline at the highest concentration of Y-27632 used. These were recorded as 100% relaxation.

To further evaluate NO regulation of the RhoA/ROK pathway, denuded NR and LHR aortic rings were permeabilized with the calcium ionophore ionomycin (1.5 µM) in calcium-free PSS. CaCl2 (1.6 mM) was then added to the bath. After the contraction plateaued, rings were relaxed with S-nitroso-N-acetyl-penicillamine (SNAP, 1 nM), Y-27632 (1 µM), or a consecutive combination of the two agents.

RhoA distribution. The cellular location of RhoA is an indicator of activity. In the inactive state, RhoA is found in the cytosol. On activation, RhoA moves to the membrane (11, 22, 23). To determine the location and therefore activity of RhoA, Western blot analysis was used. Rings were hung in water-jacketed tissue baths for contractile experiments and equilibrated 60 min. After equilibration, tissues were treated with L-NNA (100 µM) or vehicle for 30 min and then exposed to the alpha 2-AR agonist UK-14304 (10 µM). Exactly 5 min after agonist stimulation (appropriate activation time determined by time course, data not shown), rings were removed from baths and frozen in liquid nitrogen. Rho distribution was measured using the method described in Sauzeau et al. (39). Briefly, frozen aorta sections were homogenated in lysis buffer containing (in mM) 20 HEPES-NaOH, 10 KCl, 10 NaCl, and 5 MgCl2 and included complete protease inhibitor (Roche; Mannheim, Germany). Homogenate was centrifuged at 13,000 g for 3 min at 4°C. The supernatant was removed and centrifuged at 100,000 g for 30 min. The supernatant (cytosolic fraction) was removed and the pellet (membrane fraction) resuspended. Protein concentration of each fraction was determined using the Bradford method (Bio-Rad).

Protein concentrations were normalized and samples loaded into a 10-20% gradient acrylamide gel for resolution with electrophoresis. Separated proteins were transferred onto polyvinylidene difluoride membranes and blocked overnight with 0.1% Tween 20, 5% milk, and 3% bovine serum albumin. Blots were incubated overnight at 4°C with 2 µg/ml anti-RhoA (1:100), followed by a secondary antibody for 1 h at room temperature (1:10,000) and were developed using enhanced chemiluminescence reagents. Blots showed more than one band; however, only one band was present at the molecular weight of RhoA. After blot analyses, blots were stained with Coomassie blue to ensure equivalent protein loading. Protein levels were compared by densitometric analysis using SigmaGel software (SPSS). Results are reported as the ratio of membrane to total RhoA.

Statistics. Data are reported as means ± SE and were analyzed with a one-way or two-way ANOVA as appropriate. Post hoc tests were performed using the Student-Newman-Keuls test. P values <= 0.05 are considered significant. Contractile studies are expressed as percent maximum tension to UK-14304. Percentages were arcsin transformed before statistical analysis to ensure normality.

Materials. The Rho kinase inhibitor Y-27632 was a generous gift from Mitsubishi Pharma (Osaka, Japan). RhoA antibodies were purchased from Santa Cruz Biotechnology. Chemiluminescent reagents were purchased from Amersham. Ionomycin was purchased from Calbiochem and SNAP was purchased from Sigma-Aldrich.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

Acute NOS inhibition does not account for increased alpha 2-AR vasoreactivity in NOS-inhibited hypertensive rats. To determine whether inhibition of NOS alone could account for increased alpha 2-AR sensitivity associated with chronic NOS inhibition hypertension, NR and LHR thoracic aorta were exposed to cumulative concentrations of the alpha 2-AR agonist UK-14304. Both endothelium-intact and endothelium-denuded preparations were tested in the presence and absence of L-NNA (100 µM). L-NNA was used in endothelium-denuded preparations to look for nonspecific effects. All LHR rings exhibited increased reactivity to UK-14304 compared with aorta from NR controls (Fig. 1). The presence of endothelium in the absence of L-NNA in both groups attenuated maximum tension. As expected, endothelium increased the EC50 for UK-14304 in NR compared with all other treatment groups. The EC50 for UK-14304 in LHR rings was similar for all treatments and was significantly lower than NR rings for all treatments (Table 1).


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  		Fig. 1.   Chronic nitric oxide (NO) synthase (NOS) inhibition hypertension, not just acute NOS inhibition, enhances sensitivity to the alpha 2-adrenergic receptor (alpha 2-AR) agonist UK-14304. Endothelium-intact (A and B) and -denuded (C and D) normotensive rats (NR) and chronic NOS-inhibited hypertensive rats (LHR) aorta rings were exposed to cumulative concentrations of UK-14304 (10-9 to 10-5 M) in the presence (B and D) and absence (A and C) of Nomega -nitro-L-arginine (L-NNA, 100 µM). To determine whether NOS inhibition alone is responsible for augmented alpha 2-AR contractile sensitivity in LHR aorta, NR aorta were exposed to cumulative concentrations of UK-14304 with and without acute NOS inhibition. LHR aorta were exposed to the same treatments as NR aorta as a control. Additionally, endothelium-denuded rings were treated with L-NNA to look for potential nonspecific effects of L-NNA. *Significantly different from NR. n, Number of animals.


                              
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  		Table 1.   UK-14304 contraction and calculated Y-27632 IC50 values in UK-14304 precontracted rings

Rho and ROK participate in alpha 2-AR contraction. To determine whether ROK plays a role in alpha 2-AR contraction, consecutive UK-14304 concentration-response curves were generated in endothelium-denuded thoracic aorta in the presence of increasing doses of Y-27632 (0, 1, and 10 µM). In both NR and LHR aorta, Y-27632 significantly and dose dependently attenuated the UK-14304 contraction (Fig. 2). Moreover, the highest dose of Y-27632 used (10 µM) completely eliminated contraction in both groups, indicating a necessary role of ROK in alpha 2-AR contraction. However, Y-27632 (1 µM) attenuated contraction more in NR aorta than in LHR aorta.


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  		Fig. 2.   Rho-associated kinase (ROK) inhibition concentration dependently attenuates alpha 2-AR contraction in NR and LHR aorta. To evaluate the contribution of ROK to the alpha 2-AR contraction, endothelium-denuded NR (A) and LHR (B) thoracic aorta rings were exposed to cumulative concentrations of the alpha 2-AR agonist UK-14304 (10-9 to 10-5 M) in the presence of vehicle or increasing concentrations of the ROK inhibitor Y-27632 (1 and 10 µM). *Significantly different from vehicle. #Significantly different from NR. n, number of animals.

Acute NOS inhibition augments Y-27632 relaxation of UK-14304 preconstricted aorta. The data from Fig. 2 suggest that ROK is involved in alpha 2-AR contraction and that Y-27632 attenuates UK-14304 contraction more in NR than in LHR aorta. To confirm these results and further evaluate the effect of chronic NOS inhibition hypertension on alpha 2-AR requirement for ROK activity, endothelium-intact and -denuded NR and LHR aorta in the presence and absence of L-NNA (100 µM) were preconstricted with UK-14304 and relaxed with Y-27632 (Fig. 3, A and B). Denuded and L-NNA-treated intact NR aorta were less sensitive to Y-27632 relaxation of UK-14304 contraction than vehicle-treated intact NR aorta. There were no differences in relaxation between treatments of LHR aorta. LHR aorta were slightly more responsive to Y-27632 than endothelium-intact, vehicle-treated NR aorta (Fig. 3C), and the calculated IC50 was decreased (Table 1). Similar to results in Fig. 2, LHR aorta were less responsive than endothelium-denuded NR aorta (Fig. 3D), and the IC50 was greater (Table 1). Although the endothelium-intact NR aorta met the viability standard (phenylephrine contraction: NR intact, 1,052 ± 44 mg; NR denuded, 1,300 ± 58 mg; LHR intact, 1,232 ± 122 mg; and LHR denuded, 1,300 ± 48 mg force), alpha 2-AR contraction in endothelium-intact NR aorta is very small, and relaxation responses in these arteries are difficult to interpret on their own. Therefore, Western blot analysis of RhoA cellular distribution was used.


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  		Fig. 3.   ROK inhibitor Y-27632 relaxed UK-14304 contracted aorta. To evaluate potential changes in ROK contribution to alpha 2-AR contraction with chronic NOS inhibition hypertension, endothelium-intact and -denuded NR and LHR thoracic aorta were exposed to cumulative concentrations of UK-14304 (10-9 to 10-5 M) in the presence of vehicle or L-NNA (100 µM). After contraction plateau, rings were exposed to cumulative concentrations of Y-27632 (0.1, 0.5, 1, and 3 µM). Relaxation to Y-27632 following UK-14304 contraction was greater in denuded and intact L-NNA-treated NR aorta than in intact vehicle-treated NR aorta (A). Additionally, denuded NR aorta (C) were more sensitive to Y-27632 than were denuded LHR (D). *Significantly different from intact vehicle-treated rings. n, number of animals.

RhoA activity is enhanced following NOS inhibition. To confirm the data in Fig. 4, Western blots were used. When activated, RhoA translocates from the cytosol to the membrane (11, 22, 23). Therefore, the presence of RhoA in the membrane fraction can be used to evaluate RhoA activity. Under basal conditions, acute NOS inhibition increased the amount of RhoA in the membrane fraction. RhoA was present in the membrane fraction from endothelium-denuded and -intact, L-NNA-treated NR aorta compared with endothelium-intact, vehicle-treated NR aorta (Fig. 4). However, basally, the amount of RhoA present in the membrane fraction in LHR aorta was not different from that in intact, vehicle-treated NR aorta. UK-14304 stimulated RhoA translocation to the membrane fraction in all groups except intact, vehicle-treated NR aorta. There were no differences between endothelium-denuded, vehicle-treated and L-NNA-treated NR aorta. Therefore, only vehicle-treated data are shown. These data suggest that acute NOS inhibition augments both basal and UK-14304-stimulated Rho activation, but chronic NOS inhibition only enhances UK-14304-stimulated RhoA activation.


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  		Fig. 4.   Basal RhoA localization in the membrane was greater in denuded and intact L-NNA-treated NR aorta than in intact vehicle-treated NR or LHR aorta. UK-14304 stimulated RhoA translocation in all groups except intact, vehicle-treated NR aorta. Endothelium-intact and -denuded NR and LHR aorta were exposed to vehicle or UK-14304 (10 µM) in the presence of L-NNA (100 µM) or vehicle. Blots with cytosolic and membrane fractions of aorta homogenates were exposed to RhoA antibody and densitometric analysis performed. A: ratio of membrane fraction to total RhoA. Stacked bars are basal (bottom) and UK-14304-stimulated (top) membrane-to-total RhoA ratio. B: representative blots of basal and UK-14304-stimulated membrane and cytosolic fractions. No differences were noted in LHR endothelium-intact and -denuded treatments; therefore, these data have been combined. *Significantly different from intact vehicle-treated NR. #Significantly different from basal. n, number of animals.

To determine whether measured RhoA was potentially affected by the amount of RhoA expressed in NR and LHR aorta, membrane and cytosolic RhoA fractions were added to determine total RhoA protein present, normalized to total protein loaded. Total RhoA was not different between LHR and NR aorta (NR: 1.89 ± 0.44; LHR: 1.64 ± 0.42).

Sensitivity to NO donors in ionomycin-permeabilized aorta. To further evaluate the potential difference in sensitivity of the RhoA/ROK pathway and NO regulation of RhoA/ROK, the NO donor SNAP was used. Along with inhibition of RhoA, NO causes vasodilation through many mechanisms, including sequestration of calcium in intracellular stores, inactivation of L-type calcium channels, and activation of calcium-sensitive potassium channels (17). To evaluate the specific effect of NO on RhoA, we used denuded aortic rings permeabilized with the calcium ionophore ionomycin (1.5 µM) and contracted with CaCl2 (1.6 mM) (Fig. 5). In this preparation, NO modulation of calcium sequestration and influx is minimal so that an effect on RhoA/ROK would be more easily observed. CaCl2 contracted LHR aorta more than NR aorta (NR: 938 ± 77, LHR: 1,240 ± 89 mg). The NO donor SNAP (1 nM) relaxed NR rings more than LHR rings. Similarly, NR rings relaxed more than LHR rings to Y-27632 (1 µM) alone. The addition of both Y-27632 and SNAP resulted in additional relaxation in both groups, but again, NR rings relaxed more than LHR rings. These data indicate that in the absence of agonist stimulation, RhoA/ROK contributes less to contraction in LHR aorta.


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  		Fig. 5.   Relaxation to S-nitroso-N-acetyl-penicillamine (SNAP) and Y-27632 in denuded ionomycin-permeabilized aortic rings was greater in NR than in LHR. Denuded aortic rings from NR and LHR were permeabilized with the calcium ionophore ionomycin (1.5 µM) and exposed to 1.6 mM CaCl2. The contraction plateaued, and SNAP (1 nM) or Y-27632 (1 µM) was added to the bath. After relaxation plateaued, Y-27632 (1 µM) or SNAP (1 nM) was added to the baths containing SNAP and Y-27632, respectively, to evaluate cumulative relaxation. NR tissues relaxed more to SNAP, Y-27632, and the combination of the two agents than did LHR tissues. Data are reported as percent relaxation to CaCl2 contraction. *Significantly different from NR. n, number of animals.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

We hypothesized that ROK participates in alpha 2-AR vascular smooth muscle contraction and that enhanced alpha 2-AR vasoreactivity with chronic NOS inhibition hypertension results from increased RhoA and ROK activity. These hypotheses were tested by using aorta from normotensive and chronic NOS-inhibited hypertensive rats. To account for the possibility that acute NOS inhibition can alter RhoA and ROK activity, NR aorta were tested with and without acute NOS inhibition. We noted that acute and chronic NOS inhibition enhanced alpha 2-AR vasoreactivity but that acute NOS inhibition alone could not account for augmented alpha 2-AR sensitivity in aorta from chronic NOS-inhibited hypertensive rats. Additionally, we demonstrated that alpha 2-AR contraction in LHR and NR thoracic aorta requires ROK activity. We found that acute and chronic NOS inhibition augmented sensitivity to Y-27632 relaxation in UK-14304-constricted aorta. However, acute NOS inhibition increased Y-27632 sensitivity more than chronic NOS inhibition hypertension. Western blots demonstrated that alpha 2-AR stimulated RhoA translocation to the membrane fraction following acute and chronic NOS inhibition, but only acute NOS inhibition augmented RhoA present in the membrane fraction in unstimulated arteries. Together, these studies indicate that alpha 2-AR contraction depends on ROK activity, but increased alpha 2-AR vasoreactivity in denuded LHR aorta is not due to enhanced RhoA or ROK activity. Also, acute NOS inhibition increases basal and alpha 2-AR stimulated RhoA and ROK activity; however, chronic NOS inhibition hypertension only enhances alpha 2-AR-stimulated and not basal RhoA activity. The confirmation of this finding is NR-denuded ionomycin-permeabilized rings relaxed more to SNAP and to Y-27632 than LHR rings, indicating that the NO-RhoA/ROK pathway is less active in chronic NOS-inhibited hypertensive aorta.

Although alpha 2-AR do not play a large role in normotensive norepinephrine contraction in vivo (10), there is an increased role in hypertension (20), making the alpha 2-AR contractile pathway intriguing. Contraction by alpha 2-AR agonists requires extracellular calcium entry through nifedipine-sensitive calcium channels (1, 26, 29) and requires tyrosine kinase(s) (19), probably to regulate intracellular calcium concentration (unpublished observations). Additionally, we have recently demonstrated a calcium-sensitivity component in alpha 2-AR contraction (unpublished observations), which suggests augmented signaling by a kinase affects calcium sensitivity. The potential role of RhoA and ROK in alpha 2-AR contraction is suggested by studies in other systems. For example, norepinephrine contraction in human small omental arteries and rabbit aorta is sensitive to inhibition by Y-27632 (27) and C3 botulinum toxin (24), respectively, suggesting that RhoA and ROK contribute to adrenergic arterial contraction. Carbachol-induced actin formation in human airway smooth muscle requires Gi activation of RhoA (39). We demonstrated that RhoA was translocated to the membrane following UK-14304 stimulation, indicating that it is activated by the alpha 2-AR Gi pathway in vascular smooth muscle. Additionally, the ROK inhibitor Y-27632 completely eliminated the consequent contraction. Together, these observations suggest the vascular smooth muscle alpha 2-AR pathway proceeds through RhoA and ROK stimulation to inactivate MLC phosphatase and induce contraction.

RhoA activation can be regulated by many pathways (38). NO, a potent vasodilator, relaxes endothelin-1 and U-46619 contraction in rabbit thoracic aorta without changing calcium levels, suggesting NO affects calcium sensitivity (5). It has been suggested that NO calcium-independent relaxation is through PKG inhibition of RhoA (37), and recent evidence indicates that NO acts as a vasodilator in part through inhibition of RhoA/ROK (8). This hypothesis is supported by the current data. We demonstrated that NOS inhibition by endothelium denuding, treatment with L-NNA in aorta, or by chronic L-NNA treatment in the animal results in enhanced UK-14304-stimulated RhoA translocation to the membrane, indicative of enhanced RhoA activity. Also, acute NOS inhibition increased RhoA in the membrane fraction basally. Similarly, the IC50 for Y-27632 attenuation of UK-14304 contraction was significantly decreased after acute and chronic NOS inhibition. These data suggest that endogenous NO inhibits RhoA and ROK activity.

The addition of a submaximal concentration of NO donor SNAP to denuded, permeabilized aortic rings contracted with CaCl2 resulted in relaxation in both NR and LHR rings. This relaxation was potentiated by Y-27632, confirming that NO relaxes arteries in part through inhibition of RhoA/ROK. However, these experiments do not exclude the possibility that SNAP and Y-27632 act in a cumulative way.

NO regulation of contraction through RhoA inhibition could be significant in hypertension where endothelium-dependent dilation is impaired (12, 31). Y-27632 administered in vivo to spontaneously hypertensive, DOCA-salt, and renal-hypertensive rats significantly and dose dependently reduced blood pressure in hypertensive animals only, suggesting that ROK may contribute to hypertension (40). The results presented here suggest that although both acute and chronic NOS inhibition enhance UK-14304-stimulated RhoA and ROK activity, acute NOS inhibition enhanced basal and UK-14304-stimulated RhoA and ROK activity more than chronic NOS inhibition.

This difference between the effect of chronic and acute NOS inhibition on RhoA and ROK has not been reported to date but is indirectly supported by the literature and previous work in our laboratory. RhoA tends to be activated during receptor-dependent vasoconstriction but not during receptor-independent, KCl contraction (42). If RhoA contributed more to alpha 2-AR contraction in denuded LHR than in denuded NR aortic rings, we would expect that KCl contraction would be similar in LHR and NR. However, we have previously shown that KCl contraction is augmented in denuded LHR aortic rings (20). This previous work supports the current data and conclusion that enhanced RhoA and ROK activation is not responsible for increased contractile sensitivity in LHR aorta. The current data in fact suggest that some desensitization of RhoA occurs with chronic NOS inhibition.

Ionomycin-permeabilized denuded rings from NR relaxed more to SNAP and Y-27632 than did LHR rings. Additionally, potentiation of SNAP relaxation with Y-27632 was greater in NR than in LHR rings. These data support the finding that the RhoA/ROK pathway may be desensitized after chronic NOS inhibition.

It has been reported that chronic NOS inhibition results in augmented plasma vasoconstrictor levels (e.g., endothelin-1, norepinephrine, or angiotensin) (6, 25, 43). Continual stimulation of RhoA by endothelin-1 and norepinephrine could result in desensitization, which would account for the lower RhoA translocation and ROK activation in chronic NOS inhibition compared with acute NOS inhibition.

RhoA desensitization following prolonged stimulation is not without precedent. Gong et al. (13) demonstrated that acute GTPgamma S treatment stimulated RhoA activation and agonist-induced calcium sensitization, but prolonged treatment (5-16 h) decreased RhoA ADP-ribosylation. Although RhoA translocation did occur, the data indicate chronic activation can downregulate RhoA activity (13). Together with the data presented here, this suggests initial activation of RhoA by acute NOS inhibition may feed back to cause desensitization or downregulation during chronic NOS inhibition.

We have previously shown LHR to have augmented contraction to KCl in denuded aorta and to CaCl2 in ionomycin-permeabilized aorta, suggesting enhanced calcium sensitivity. Increased calcium sensitivity may result in the augmented alpha 2-AR contractile response seen in LHR aorta. Three kinases that contribute to alpha 2-AR contraction could be responsible for enhanced calcium sensitivity, ERK1/2, ROK, and PKC (2, 6, 7, 13, 18, 32). We have previously shown that although ERK1/2 participates, it does not play a larger role in LHR than in NR alpha 2-AR contraction. Similarly, the data presented here suggests that ROK is required in both LHR and NR contraction, but it is not responsible for the enhanced UK-14304 contraction in LHR. Aburto et al. (1) showed that PKC contributes to alpha 2-AR contraction in the rat aorta. Additionally, calcium-sensitive PKC-alpha activity is increased by NOS inhibition in pregnant rats (21). It is possible that there is either enhanced PKC activity or a change in PKC isoform activated after chronic NOS inhibition, which augments alpha 2-AR contraction. Current work in our laboratory is investigating this possibility.

The aim of the current study was to evaluate the contribution of RhoA and ROK to alpha 2-AR contraction in NR and LHR aorta and to determine whether augmented RhoA and ROK activities were responsible for enhanced alpha 2-AR contractile sensitivity in denuded LHR aorta. Although we demonstrated that ROK is apparently not responsible for enhanced alpha 2-AR sensitivity in LHR aorta, there is currently not an assay for direct measurement of ROK activity. Future experiments in this area with such an assay are needed to confirm our present conclusions. This study demonstrated that RhoA and ROK contribute to alpha 2-AR contraction and that this contribution is changed with chronic NOS inhibition hypertension. These data provide convincing evidence that endogenous NO regulates RhoA and imply NO regulation of vascular smooth muscle tone in vivo depends in part on RhoA.


    ACKNOWLEDGEMENTS

We thank Pam Allgood and Marina Martinez for expert technical assistance.


    FOOTNOTES

This study was supported by an Atorvastatin Research Award and National Heart, Lung, and Blood Institute Grant 03852 (to N. L. Kanagy).

Address for reprint requests and other correspondence: R. W. Carter, 915 Camino de Salud, Vascular Physiology Research Division, Dept. of Cell Biology and Physiology, Univ. of New Mexico Health Sciences Center, Albuquerque, NM 87131-5218 (E-mail: bcarter{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.

May 30, 2002;10.1152/ajpheart.01101.2001

Received 14 December 2001; accepted in final form 23 May 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
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