Published online before print February 19, 2007, doi: 10.1161/01.HYP.0000259669.40991.1e
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(Hypertension. 2007;49:893.)
© 2007 American Heart Association, Inc.
Original Articles |
Novel Nitric Oxide Synthase–Dependent Mechanism of Vasorelaxation in Small Arteries From Hypertensive Rats
From the Vascular Biology Center, Medical College of Georgia, Augusta.
Correspondence to Jennifer S. Pollock, Vascular Biology Center, CB 3213, Medical College of Georgia, Augusta, GA 30912. E-mail jpollock{at}mcg.edu
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Abstract |
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To determine the mechanism(s) involved in vasorelaxation of small arteries from hypertensive rats, normotensive (NORM), angiotensin II-infused (ANG), high-salt (HS), ANG high-salt (ANG/HS), placebo, and deoxycorticosterone acetate-salt rats were studied. Third-order mesenteric arteries from ANG or ANG/HS displayed decreased sensitivity to acetylcholine (ACh)-induced vasorelaxation compared with NORM or HS, respectively. Maximal relaxations were comparable between groups. Blockade of Ca2+-activated K+ channels had no effect on ANG versus blunting relaxation in NORM (log EC50: –6.8±0.1 versus –7.2±0.1 mol/L). NO synthase (NOS) inhibition abolished ACh-mediated relaxation in small arteries from ANG, ANG/HS, and deoxycorticosterone acetate-salt versus blunting relaxation in NORM, HS, and placebo (% maximal relaxation: ANG: 2.7±1.8; ANG/HS: 7.2±3.2; NORM: 91±3.1; HS: 82.1±13.3; deoxycorticosterone acetate-salt: 35.2±17.7; placebo: 79.3±10.3), indicating that NOS is the primary vasorelaxation pathway in these arteries from hypertensive rats. We hypothesized that NO/cGMP signaling and NOS-dependent H2O2 maintains vasorelaxation in small arteries from ANG. ACh increased NOS-dependent cGMP production, indicating that NO/cGMP signaling is present in small arteries from ANG (55.7±6.9 versus 30.5±5.1 pmol/mg), and ACh stimulated NOS-dependent H2O2 production (ACh: 2.8±0.2 µmol/mg; N
-nitro-L-arginine methyl ester hydrochloride+ACh: 1.8±0.1 µmol/mg) in small arteries from ANG. H2O2 induced vasorelaxation and catalase blunted ACh-mediated vasorelaxation. In conclusion, Ca2+-activated K+ channel–mediated relaxation is dysfunctional in small mesenteric arteries from hypertensive rats, and the NOS pathway compensates to maintain vasorelaxation in these arteries through NOS-mediated cGMP and H2O2 production.
Key Words: NO • hydrogen peroxide • hypertension • endothelium-dependent vasorelaxation • mesenteric arteries • aorta
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Introduction |
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Hypertension is associated with impaired endothelium-dependent vasorelaxation in response to stimuli.1 Acetylcholine (ACh)-induced vasorelaxation is blunted in conduit vessels from genetic and experimental hypertensive rodent models such as spontaneously hypertensive (SHR),2 deoxycorticosterone acetate (DOCA) salt-induced hypertensive,3 renovascular hypertensive,4 and angiotensin II–infused (ANG) hypertensive rats.5 Endothelial dysfunction has been associated with reduced NO bioavailability.6 However, both unchanged and impaired ACh-induced vasorelaxation have been observed in small mesenteric arteries from DOCA-salt,7,8 SHR,9,10 and ANG rats11 and mice.12 Therefore, NO bioavailability in resistance arteries may be different than that observed in conduit vessels.
Small resistance arteries are physiologically critical for the regulation of blood pressure and local blood flow. Endothelial cells release endothelium-derived relaxing factors (EDRFs) in response to stimuli. It is known that small resistance arteries produce >1 EDRF, including NO, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). Endothelial NO synthase (NOS3) produces NO when activated by neurohumoral or mechanical stimuli, which stimulate soluble guanylyl cyclase to produce cGMP.13 In addition, NOS3 enzymatic activity has been shown to become uncoupled and generate superoxide (O2–).14 Many models of hypertension have documented increased vascular O2– production from various enzymatic sources, including uncoupled NO synthase (NOS).15 Increased vascular O2– generation may limit the amount of bioavailable NO and increase hydrogen peroxide (H2O2) through the action of superoxide dismutase (SOD). H2O2 has been reported to induce contraction and/or relaxation in vascular tissue, dependent on the species, vascular bed, and experimental conditions. H2O2 has been shown to cause contraction in aorta, pulmonary artery, and superior mesenteric artery of the rat, the porcine pulmonary artery, and the canine basilar artery (for review, see Reference 16). H2O2 also mediates endothelium-dependent and -independent vasorelaxation in mouse, rat, and human mesenteric arteries and porcine, canine, and human coronary microvessels.16–18
EDHF has been described as a principal mediator of endothelium-dependent vasorelaxation in small resistance arteries from normotensive animals (for review, see References 19 and 20). EDHF appears to be the dominant endothelium-dependent vasorelaxation pathway when the endothelial NOS/NO pathway is absent as demonstrated in NOS3 knockout mice.21 Yet, studies have shown that the EDHF pathway is dysfunctional in experimental models of hypertension with reduced NO bioavailability.22 Thus, it is unclear which EDRF pathway is active in small arteries of animal models of hypertension.
The initial aims of the present study were to determine whether small mesenteric arteries and aorta from hypertensive rats display blunted vasorelaxation and to evaluate the relative participation of the NOS pathway, cyclooxygenase pathway, and Ca2+-activated K+ channel to mediate relaxation in small arteries from hypertensive rats in comparison with normotensive rats. These experiments demonstrated that small arteries and the aorta from hypertensive rats have impaired ACh-induced vasorelaxation. As expected, the NOS pathway in the aorta from hypertensive rats was blunted. In contrast, the NOS pathway was the predominant active pathway for ACh-induced vasorelaxation in small arteries from hypertensive rats. These results led us to design further experiments to test the hypothesis that both NO and H2O2 serve as NOS-dependent ACh-induced mediators from small mesenteric arteries of hypertensive rats.
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Methods |
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Animals
Male Sprague–Dawley rats (200 to 250 g; Charles River Laboratories, Wilmington, Mass, or Harlan Laboratories, Prattville, Ala) were divided into 6 groups: normotensive rats on regular chow (NORM); angiotensin II–infused hypertensive rats on regular chow (ANG); NORM on high-salt diet (diet containing 8% NaCl; Harlan-Teklad, Madison, Wis; HS); ANG on high-salt diet (ANG/HS); uninephrectomized, placebo pellet-implanted rats (placebo); and uninephrectomized, deoxycorticosterone acetate pellet-implanted hypertensive rats (DOCA). ANG and DOCA hypertension were established as described previously.23,24 The ANG model of hypertension is widely used to model human angiotensin II–dependent hypertension. The ANG/HS and DOCA models of hypertension demonstrate severe vascular remodeling and inflammation. Systolic blood pressure was measured by tail-cuff plethysmography. After 2 weeks, rats were anesthetized with sodium pentobarbital (Nembutal, 50 mg/kg IP; Abbott), and the aorta and mesenteric arteries were isolated. Animal protocols were approved and monitored by the Medical College of Georgia Institutional Animal Care and Use Committee.
Isolated Artery Preparation and Vascular Reactivity Protocol
Aortic rings and third-order branches of the superior mesenteric artery were isolated and mounted on wire myographs (Danish Myo Technology A/S) containing physiological saline solution as described previously.25 In some artery segments, mechanical rubbing of the luminal surface with human hair denuded the endothelial layer, which was confirmed by absence of ACh-induced vasorelaxation after preconstriction. Cumulative concentration–response curves to ACh (1 nmol/L to 10 µmol/L) were performed after precontraction of arterial segments. Artery segments from DOCA or placebo were precontracted with phenylephrine (1 µmol/L); all others were precontracted with U-46619 (1 µmol/L). To study the participation of vasorelaxation pathways, Ach concentration–response curves were performed in the presence and absence of the following: (1) NOS inhibitor, N-nitro-L-arginine methyl ester hydrochloride (L-NAME; 100 µmol/L); (2) cyclooxygenase inhibitor, indomethacin (Indo; 10 µmol/L); (3) Ca2+-activated K+ channel blockers, charybdotoxin (ChTX; 50 nmol/L) plus apamin (Ap; 50 nmol/L); (4) nonspecific K+ channel blocker, tetraethylammonium (1 mmol/L); (5) catalase (5000 U/mL); (6) various combinations of inhibitors; or (7) vehicle (physiological saline solution as control). Cumulative concentration–response curves were also performed with sodium nitroprusside (SNP; 100 pmol/L to 10 µmol/L) in the presence of L-NAME (100 µmol/L) and with H2O2 (10 nmol/L to 1 mmol/L) in the presence and absence of L-NAME (100 µmol/L) or endothelium-denuded artery segments. All of the inhibitors were preincubated for 30 minutes before experiments, except for catalase, which was preincubated for 1 hour. The degrees of precontraction of artery segments with all of the treatments were similar to the control. Relaxation responses to ACh were plotted as a percentage of relaxation from the maximum contraction. Each experiment was conducted on artery segments from different animals.
Measurement of Intracellular cGMP Content
Aorta and mesenteric arterial bed (excluding the superior mesenteric artery) were isolated as described previously,26 separated into 4 sections, and incubated at 37°C in oxygenated physiological saline solution with 3-isobutyl-1-methylxanthine (300 µmol/L) for 15 minutes in the presence and absence of L-NAME (100 µmol/L) or L-NAME plus SNP (10 µmol/L). Additional arterial segments were preincubated with L-NAME for 5 minutes before the addition of ACh (1 µmol/L) for 10 minutes. cGMP was extracted as described previously, quantitated by radioimmunoassay, and normalized to milligrams of protein content.27 Protein concentrations were determined by standard Bradford assay (Bio-Rad Laboratories) using bovine serum albumin as the standard.
Measurement of Vascular H2O2 Production
Aorta and mesenteric bed (excluding the superior mesenteric artery) were isolated, separated into 4 sections, and placed into modified Krebs/HEPES buffer (mmol/L: 99.01 NaCl, 4.69 KCl, 1.03 KH2PO4, 1.20 MgSO4 7H2O, 25.0 NaHCO3, 5.6 dextrose, 20.0 Na-HEPES, and 2.50 CaCl2). The amplex red assay (a fluorometric horseradish peroxidase–linked assay, Molecular Probes) was used to quantitate H2O2 as described previously.28 Briefly, arterial segments were incubated with amplex red reagent and horseradish peroxidase in modified Krebs/HEPES buffer protected from light at 37°C for 60 minutes in the presence and absence of L-NAME (100 µmol/L), ACh (1 µmol/L), or L-NAME plus ACh. H2O2 production was normalized to milligrams of protein content.
Materials
Angiotensin II was purchased from Phoenix Pharmaceuticals, Inc. Miniosmotic pumps were purchased from Alzet. Deoxycorticosterone acetate pellet and placebo pellet were purchased from Innovative Research of America, and catalase was purchased from Roche Applied Science. All of the other chemicals were purchased from Sigma-Aldrich.
Data Analysis
Values are expressed as mean±SEM. The concentration–response curves to ACh were analyzed using nonlinear regression of sigmoidal concentration–response curves (GraphPad Prism), which were used to calculate the EC50. Blood pressure values and vascular reactivity measurements were compared using Student’s t test for paired comparisons. All of the other values were analyzed by ANOVA followed by a Fisher least significant difference posthoc test for multiple comparisons (Statistica). A value of P<0.05 was considered statistically significant.
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Results |
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Systolic Blood Pressure
Systolic blood pressure was significantly increased in ANG and ANG/HS compared with NORM and HS, respectively (ANG: 197±3 mm Hg, P<0.0001, n=64; NORM: 123±2 mm Hg, n=54; ANG/HS: 202±5 mm Hg, P<0.0001, n=11; HS: 129±4 mm Hg, n=7). DOCA were also confirmed to have significantly higher systolic blood pressure over placebo (DOCA: 197±2 mm Hg, P<0.0001, n=7; placebo: 135±4 mm Hg, n=9). Increased mean arterial pressure has been documented by telemetry in these models of hypertension.29,30
ACh-Induced Vasorelaxation
To evaluate the degree of vasorelaxation in third-order mesenteric arteries and aortic rings from ANG hypertensive rats, cumulative concentration–response curves to ACh were performed. Third-order mesenteric arteries from ANG are less sensitive to ACh compared with arteries from NORM (P=0.015); however, no difference in the maximum response to ACh was detected between groups (Figure 1A; Table 1). In aorta, maximal relaxation to ACh was blunted in ANG compared with NORM (P=0.001; Figure 1B; Table 1). The cumulative concentration–responses to SNP were comparable between all of the experimental and control groups studied (Table 2).
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Significant difference in maximum response to ACh versus NORM (P<0.05).
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To evaluate the participation of the cyclooxygenase pathway, Ca2+-activated K+ channels, and the NOS pathway to mediate vasorelaxation in small arteries from ANG and NORM, arteries were incubated with pharmacological blockers before initiation of the concentration–response curves to ACh (Figure 2A and 2B; Table 1). No change in vasorelaxation in small arteries from either NORM or ANG was detected with Indo pretreatment. ChTX+Ap significantly decreased sensitivity to ACh in small arteries from NORM (P=0.009), whereas these blockers did not alter vasorelaxation in arteries from ANG. Similarly, pretreatment with tetraethylammonium significantly decreased sensitivity in small arteries from NORM (P=0.011), whereas no change was detected in the small arteries from ANG. L-NAME significantly decreased sensitivity in small arteries from NORM (P=0.0005) and abolished the ACh-induced relaxation in ANG.
Isolated arteries were incubated with a combination of the pharmacological blockers before initiation of the concentration–response curves to ACh to verify that blockade of all pathways abolishes the vasorelaxation (Figure 2C and 2D; Table 1). Pretreatment with Indo and ChTX+Ap significantly decreased the sensitivity to ACh in NORM (P=0.010), whereas no change was noted in ANG. Indo and L-NAME also decreased ACh sensitivity in NORM (P=0.0004; Figure 2C), whereas this pretreatment completely abolished the vasorelaxation in ANG (Figure 2D). Pretreatment of small mesenteric arteries with all of the inhibitors abolished the ACh-induced vasorelaxation in both NORM and ANG (Figure 2C and 2D).
The ANG/HS and DOCA models of hypertension were used as models of severe vascular remodeling and inflammation, as well as to determine whether this phenomenon depends on angiotensin II. Small arteries from ANG/HS displayed an impaired ACh-induced vasorelaxation response when compared with HS (P=0.0001; Table 1), and small arteries from DOCA showed a nonsignificant tendency to decrease sensitivity of ACh-induced vasorelaxation compared with arteries from placebo (P=0.074; Table 1). The contribution of the NOS pathway to ACh-induced vasorelaxation in small arteries from HS, ANG/HS, placebo, and DOCA was also determined (Figure 3; Table 1). L-NAME decreased sensitivity to ACh in small arteries from HS (P=0.0001) and placebo (P=0.037) rats and largely abolished the vasorelaxation response in arteries from ANG/HS and DOCA. Thus, these experiments indicate that the NOS pathway is the primary vasodilating pathway present in small arteries from these 3 models of hypertension and is angiotensin II independent.
NO/cGMP in Small Mesenteric Arteries and Aorta
NO production in small mesenteric arteries and aortic rings was determined by measuring the cGMP content in the presence and absence of L-NAME and/or ACh (Figure 4). The basal cGMP content in small mesenteric arteries from ANG was significantly less compared with arteries from NORM (P<0.0001; Figure 4A). ACh stimulation significantly increased cGMP production compared with basal cGMP in small mesenteric arteries from NORM (P=0.019) and ANG (P=0.055). As shown in Figure 4B, basal cGMP production in aortic rings from ANG was significantly less when compared with aortic rings from NORM (P=0.0004). ACh induced a significant increase in cGMP production compared with basal in aortic rings from NORM (P=0.0003), whereas ACh stimulation did not increase cGMP production when compared with basal cGMP of aortic rings from ANG. L-NAME abolished the production of cGMP in all of the groups studied (Figure 4A and 4B). SNP-induced cGMP production was comparable in both small mesenteric arteries and aortic rings from both NORM and ANG (Table 2).
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H2O2-Induced Vasorelaxation
The results in Figure 4A demonstrated that there was a significant increase in ACh-induced cGMP production in small arteries from ANG, although not to the same plateau as in NORM despite maximal relaxation. Thus, we reasoned that another NOS-dependent EDRF, possibly H2O2, may be present in small arteries from hypertensive rats. To test the involvement of H2O2 in ACh-induced vasorelaxation, small mesenteric arteries from ANG were pretreated with catalase for 1 hour, and cumulative concentration–response curves to ACh were evaluated (Figure 5A). Catalase treatment blunted the ACh-induced vasorelaxation in small arteries from ANG (log EC50 [mol/L], control: –6.8±0.1 versus catalase: –6.1±0.2; P=0.003; n=6). To confirm the capacity of H2O2 to mediate vasorelaxation in small mesenteric arteries from ANG, cumulative concentration–response curves to H2O2 were performed with control endothelium-intact, L-NAME–treated endothelium-intact, and endothelium-denuded small mesenteric artery segments (Figure 5B). H2O2 induced vasorelaxation of preconstricted small arteries from ANG. Preincubation with L-NAME or denudation of endothelium significantly decreased sensitivity to H2O2 in small arteries from ANG (log EC50 [mol/L], control: –4.7±0.02, n=7, versus L-NAME: –4.4±0.03; P<0.001; n=8; denudation: –4.4±0.05; P=0.0003; n=7).
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NOS-Dependent H2O2 Production in Small Mesenteric Arteries and Aorta
Basal and ACh-stimulated H2O2 production were determined in the presence and absence of L-NAME in small arteries and aortic rings from both NORM and ANG (Figure 6). As shown in Figure 6A, basal and ACh-stimulated H2O2 production were similar in small mesenteric arteries from NORM. Basal H2O2 production was significantly increased in small arteries from ANG when compared with NORM (P=0.047). ACh stimulation in small arteries from ANG significantly increased H2O2 levels when compared with the basal H2O2 production (P=0.015). Interestingly, L-NAME significantly decreased the ACh-stimulated H2O2 production in small arteries from ANG (P=0.0002). In aortic rings, basal H2O2 production was increased in ANG compared with NORM (P=0.0001); however, ACh stimulation did not change the H2O2 production in either NORM or ANG (Figure 6B). H2O2 production in aortic rings from both NORM and ANG was L-NAME independent.
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Discussion |
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The novel findings of the present study are as follows: (1) small mesenteric arteries from 3 models of hypertension all demonstrate the maintenance of maximal ACh-induced vasorelaxation similar to normotensive controls; (2) the NOS-dependent component of the vasorelaxation is dramatically increased in small arteries from hypertensive rats; and (3) the increased NOS-dependent component of the ACh-induced vasorelaxation in small arteries from hypertensive rats is mediated by NOS-derived NO/cGMP, as well as NOS-dependent H2O2. Thus, the NOS pathway is the primary EDRF pathway in small mesenteric arteries from experimental models of hypertension. These findings reveal a novel regulatory mechanism of the NOS pathway in small arteries distinct from large arteries. NOS-mediated H2O2 is a component of the ACh-induced vasorelaxation, which we suggest compensates for reduced NO bioavailability. This study also confirmed previous reports that the Ca2+-activated K+ channel–sensitive component of vasorelaxation in small arteries from hypertensive rats is dysfunctional and that impaired NOS-dependent cGMP production is responsible for the blunted aortic vasorelaxation in hypertensive rats.
Endothelial Dysfunction in Large and Small Arteries Is Distinct
Conduit arteries from hypertensive models have "endothelial dysfunction" or a blunted vasorelaxation in response to endothelium-dependent agonists. These studies have led to the common dogma that endothelial dysfunction is mediated by reduced NO bioavailability and decreased activation of soluble guanylyl cyclase leading to blunted vasorelaxation. The present study confirms this mechanism in aortic rings. In contrast, small mesenteric arteries from hypertensive rats have a maximal response to ACh- and SNP-induced vasorelaxation that is comparable to arteries from normotensive rats. Therefore, small arteries have a distinct mechanism to maintain vasorelaxation during hypertension.
Small resistance arteries have multiple endothelial-dependent vasodilating mechanisms, the major ones being NOS, cyclooxygenase, and EDHF. In this study, pharmacological blockade of these pathways was used to determine the components of the ACh-induced vasorelaxation. Only the NOS and K+ channel pathways are active in small arteries of normotensive rats, and, interestingly, the data indicate that these pathways may interact. Future studies will examine this interaction. There was a loss of the K+ channel pathway in small arteries from hypertensive rats, which is consistent with previous reports in small mesenteric arteries in SHR and ANG rats.10,31–35 Studies that have assessed EDHF- and NOS-mediated dilation in mesenteric resistance arteries from hypertensive rats and mice report decreased NO and/or EDHF contributions to relaxation in mesenteric arteries.12,32–35 Although it is very difficult to compare these studies because of differences in experimental protocols and/or animal models, many of these studies observed an imbalance of the EDHF and/or NOS pathways in the vasodilator response. The present data demonstrate that there is a shift to the NOS pathway to maintain endothelium-dependent relaxation in mesenteric resistance arteries from hypertensive rats. Liu et al36 have demonstrated increased expression of the 
-subunit of Ca2+-activated K+ channel with enhanced K+ channel activity in freshly isolated aortic smooth muscle cells from SHRs. Also, the ß1 subunit of the large-conductance K+ channel expression is upregulated in mesenteric arteries from SHRs, and this is normalized with captopril treatment.37 Amberg et al38 reported that large-conductance K+ channels have a reduced ability to respond to Ca2+ sparks and modulate vascular tone, and the ß1 subunit is depressed in cerebral arteries from ANG. Therefore, the mechanism of Ca2+-activated K+ channel–mediated vasorelaxation dysfunction in small arteries is unknown, because there does not appear to be a clear correlation of large-conductance K+ channel subunit expression and functional activity in isolated arteries from models of hypertension.
NOS-Dependent NO and H2O2 Maintain Vasorelaxation in Small Arteries
We hypothesized that the NOS pathway is activated to generate both NO and H2O2 to maintain ACh-induced vasorelaxation in small mesenteric arteries from hypertensive rats. Our laboratory previously demonstrated reduced basal NO/cGMP signaling via dysfunctional NOS phosphorylation in small mesenteric arteries from DOCA rats with no increased vascular O2–.27 Interestingly, reduced phenylephrine sensitivity in small arteries from DOCA-salt hypertensive rats was normalized after combined antioxidant and L-NAME treatment but not in experiments with these reagents separately, suggesting that an NOS-derived vasodilatory reactive oxygen species, possibly H2O2, mediates this effect.
The present study confirmed our previous report that small and conduit arteries from hypertensive rats have reduced basal NO/cGMP signaling27 and shows that ACh stimulation of small arteries from both normotensive and hypertensive rats activates NOS, indicated by significant increases in cGMP production. ACh-induced cGMP production was not evident in aortas from hypertensive rats. This difference between large and small arteries was not because of a defect in the response to NO, because SNP stimulated the same level of cGMP production in both small and large arteries from normotensive and hypertensive rats. Despite comparable ACh-induced relaxation, ACh activates the NO/cGMP pathway in small mesenteric arteries even under hypertensive conditions, although to a lesser extent than from normotensive rats.
Several reports have shown a catalase-sensitive component of vasorelaxation in human, rat, and mouse small mesenteric and coronary arteries39–41; however, no reports have shown NOS-dependent H2O2 production mediating vasorelaxation. We confirmed that catalase treatment blunted ACh-induced relaxation in small arteries from ANG and verified that H2O2 directly mediates vasorelaxation. Furthermore, we found that H2O2-induced relaxation is, at least in part, endothelium and NOS dependent in small arteries from ANG.
Small mesenteric arteries and aortic rings from hypertensive rats have significantly higher levels of basal H2O2 when compared with vessels from normotensive rats. ACh-induced H2O2 production was evident only in small arteries from hypertensive rats and was L-NAME sensitive, further supporting the role of H2O2 as an EDRF. We conclude that ACh stimulates NOS-mediated cGMP and H2O2 production to maintain vasorelaxation in small arteries from hypertensive rats.
It is not clear whether the ACh-induced L-NAME-sensitive H2O2 production is directly or indirectly generated from NOS. Rosen and colleagues42–44 have shown direct production of H2O2 by purified neuronal NOS (NOS1) and inducible NOS (NOS2); however, NOS3 has not yet been studied. Purified NOS1 generates H2O2 when the NOS cofactor, tetrahydrobiopterin, is bound and L-arginine levels are low, whereas NOS1 generates O2– when tetrahydrobiopterin is not bound. Alternatively, H2O2 production may be elicited by the conversion of uncoupled NOS-mediated O2– either spontaneously or via SOD. Fukai et al45 have reported that extracellular SOD is a principal regulator of the endothelium-derived NO bioavailability in conduit arteries, and extracellular SOD has been reported to be upregulated in ANG.46 However, future studies are necessary to determine the source(s) of H2O2 in these arteries.
The mechanism of NOS-dependent H2O2-mediated vasorelaxation is not yet clear. H2O2-mediated vasorelaxation in the coronary circulation and the superior mesenteric artery depends on 4-aminopyridine–sensitive K+ channels,18,47 whereas H2O2-mediated vasorelaxation in the superior mesenteric artery from SHR depends on Ca2+-activated K+ channels.18 This is opposed to our study and others demonstrating dysfunctional K+ channel activation in arteries from hypertensive rats. It appears unlikely that Ca2+-activated K+ channels mediate the NOS-dependent H2O2-mediated vasorelaxation seen in this study. Other pathways will be required to determine the likely mechanism for the H2O2-mediated vasorelaxation. This mechanism will be complex, because we show both NOS-independent, as well as NOS-dependent, H2O2 production.
Perspectives
This study demonstrates a novel mechanism for NOS to maintain ACh-induced vasorelaxation in compensation for the dysfunctional EDHF pathway in small arteries from hypertensive rats. Our laboratory reported upregulated NOS3 expression and activity in the cytosolic compartment of mesenteric arteries from hypertensive rats,26 although NOS3 is a membrane-associated enzyme through posttranslational modifications. The physiological consequences of altered NOS3 compartmentalization are unknown, and we have hypothesized that cytosolic NOS3 is uncoupled and serves as a source of H2O2 production.
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Acknowledgments |
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We gratefully acknowledge Dr David M. Pollock for many helpful discussions and the expert technical assistance of Heather Walker.
Sources of Funding
We acknowledge funding from the National Institutes of Health (J.C.S.: AG24616; J.S.P.: HL60653 and HL69999; J.D.I./J.S.P.: HL74167; J.D.I.: HL59699) and PhRMA Foundation Predoctoral Fellowship to J.M.S. J.D.I. and J.S.P. are Established Investigators of the American Heart Association.
Disclosures
None.
Received August 4, 2006; first decision August 24, 2006; accepted January 24, 2007.
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