Redox Signaling of the Arteriolar Myogenic Response
Arteriolar vascular smooth muscle cells (VSMCs) are mechanosensitive, constricting to elevations in transmural pressure (PTM). The goal of the present study was to determine using mouse isolated tail arterioles and arteries whether oxidant signaling regulates this myogenic response. In response to PTM elevation, VSMCs of arterioles but not arteries generated constriction and increased reactive oxygen species (ROS) activity (using the H2O2-sensitive probe dichlorodihydrofluorescein). Arterioles had increased expression of NADPH oxidase components compared with arteries. Inhibition of NADPH oxidase, using mice with targeted impairment of enzyme components (p47phox or rac1) or diphenyleneiodonium, prevented the pressure-induced generation of ROS. When ROS activity was inhibited, either by inhibiting NADPH oxidase or with N-acetylcysteine, the myogenic constriction was abolished. The myogenic constriction was also inhibited by catalase, which inactivates H2O2, but was unaffected by a cell-permeant mimic of superoxide dismutase (MnTMPyP). α1-Adrenergic constriction was not associated with altered ROS activity and was not affected by inhibition of NADPH oxidase or ROS. Exogenous H2O2 constricted VSMCs of arterioles but not arteries. Thus, NADPH oxidase and ROS, in particular H2O2, contribute to the myogenic response of arteriolar VSMCs.
Vascular smooth muscle cells (VSMCs) of arterioles, but not arteries, are mechanosensitive, constricting to elevations in transmural pressure (PTM). This myogenic response contributes to blood flow autoregulation and the establishment of basal vascular tone.1 The response is an inherent property of arteriolar VSMCs involving calcium-dependent actin/myosin interaction, but the more proximal signaling components have not been clearly defined.1 In cultured cells, mechanical stress initiates integrin-dependent activation of rho GTPases (rho, rac1, and CDC42), leading to reorganization of the cytoskeleton.2,3 Cytoskeleton reorganization by rac1 is mediated by NADPH oxidase and generation of reactive oxygen species (ROS).4 This enzyme complex, comprising Nox1, p47phox, p67phox, p22phox, and rac1, is a key signaling system in cultured, noncontractile VSMCs.5,6 The aim of the present study was to determine whether the rac1/NADPH oxidase/ROS signaling pathway regulates the myogenic response of arteriolar VSMCs.
Materials and Methods
Mouse-tail arterioles and arteries were cannulated in a microperfusion chamber (Living Systems) and studied in the absence of flow as described.7 Unless stated otherwise, arterioles with intact endothelium were analyzed. Involvement of animals in the study was approved by the Ohio State University Animal Care and Use Committee.
Endothelium-denuded vessels7 were incubated with the H2O2-sensitive probe 5-(and 6)-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate (DCF), 5 μg/mL, for 30 minutes (37°C, PTM of 10 mm Hg). Because activation of DCF fluorescence is irreversible, fluorescent images (Zeiss, LSM 410) were captured at a PTM of 10 mm Hg before and after PTM had been increased (90 mm Hg, 1 minute). Maintaining vessels at 10 mm Hg did not change DCF fluorescence. Images were quantified using Metamorph software.
Transgenic mice expressing a dominant-negative mutant of human rac1 (rac-DN, cDNA; gift of Alan Hall, London, England), with threonine17 to asparagine substitution, were generated in FVB/N mice using the smooth muscle α-actin promoter (gift of Art Strauch, Ohio State University, Columbus, Ohio). The genome of the mice incorporated the cDNA of rac-DN, including its polyadenylation site. Founder mice were selected on the basis of Southern blot analysis, and one confirmed to have the highest number of human rac-DN gene copies used to establish a stable transgenic line by breeding it with nontransgenic FVB/N mates. Reverse transcriptase–polymerase chain reaction analysis confirmed expression of rac-DN in smooth muscle, including blood vessels and intestine, whereas the transcript was not detected in control mice nor in the brain, heart, liver, skeletal muscle, and testis of transgenic mice. Responses in rac-DN mice were compared with nontransgenic littermates. All other experiments were performed on C57Bl6 mice; p47phox−/− mice8 were congenic to C57Bl6.
Western Blot Analysis
Endothelium-denuded vessels from 20 mice were processed as described.7 Antibodies were β-actin (Sigma), p47phox, p67phox (Transduction Laboratories), and rac-1 (Upstate Biotechnology).
Data are expressed as mean±SEM for n number of animals. Vasomotor responses were expressed as percentage change in the basal diameter at 10 mm Hg. Statistical evaluation was by Student’s t test, except for multiple comparisons, when ANOVA followed by Scheffe’s analysis was used.
At a PTM of 10 mm Hg, the internal diameters of arterioles and arteries were 88.5±5.4 μm (n=18) and 184.4±13.4 μm, respectively (n=8). An increase in PTM (10 to 90 mm Hg) caused arterioles to dilate, then constrict and initiate vasomotion, maintaining a diameter similar to that at a PTM of 10 mm Hg (Figure 1A). The dilation represented passive opening and was dramatically increased by vasodilators (Figure 1A). Increasing PTM in arteries caused only passive dilation, and vasodilators had no effect (Figure 1A).
Elevating PTM in arterioles increased ROS activity in VSMCs (2.53±0.30-fold increase in DCF fluorescence, n=13) (Figure 1B). The basal level of ROS was reduced and the pressure-induced elevation was abolished by the antioxidant N-acetylcysteine (NAC) (20 mmol/L, data not shown) or by impairment of NADPH oxidase using arterioles from p47phox−/− or rac-DN mice or using the nonselective inhibitor diphenyleneiodonium (DPI) (1 μmol/L) (Figure 1B). In contrast, arteries had low levels of ROS activity, which did not increase after PTM elevation (Figure 1B). Arteries had decreased expression of the NADPH oxidase components rac1, p47phox, and p67phox compared with arterioles (Figure 2D).
The myogenic constriction to elevated PTM was inhibited by impairment of NADPH oxidase (rac-DN or p47phox−/− arterioles; DPI, 1 μmol/L), and only passive dilation was observed (Figures 2A and 2B). Similar results were obtained by inhibiting ROS with NAC (20 mmol/L) (Figures 3A and 3B). Inhibition of NADPH oxidase and decreased ROS activity, in particular superoxide, might depress constriction indirectly by augmenting endothelium-derived nitric oxide (NO).9 However, inhibition of NADPH oxidase (DPI; p47phox−/− or rac-DN arterioles) or ROS activity (NAC) also inhibited the myogenic response in endothelium-denuded arterioles or in the presence of NG-nitro-l-arginine methyl ester (L-NAME) (100 μmol/L), an NO synthase inhibitor (Figure 2E and data not shown). Furthermore, the myogenic constrictor response was not affected by a cell-permeant mimic of superoxide dismutase (SOD), MnTMPyP,4 which catalyzes the dismutation of superoxide to H2O2, but was abolished by catalase, which inactivates H2O2 (Figures 3A and 3B). The inhibitory effect of catalase on myogenic constriction and DCF fluorescence (Figures 1B, 3A, and 3B⇓) was maximal after 4 hours, consistent with intracellular accumulation in VSMCs.10 Exogenous H2O2 constricted endothelium-denuded arterioles but not arteries (Figure 3D).
Constriction to the α1-adrenergic agonist phenylephrine was not associated with increased ROS activity (data not shown) and was not affected by inhibition of NADPH oxidase (DPI; rac-DN or p47phox−/− arterioles) or of ROS (NAC, catalase) (Figures 2C and 3C⇑).
Elevation in PTM increased ROS activity in arteriolar VSMCs. The source of ROS was likely to be NADPH oxidase based on the reduced activity in p47phox−/− or rac-DN arterioles and the inhibitory effect of DPI. When the increase in ROS was inhibited, either by the antioxidant NAC or by inhibition of NADPH oxidase, the myogenic constriction to elevated PTM was abolished. Therefore, oxidant signaling by the rac/NADPH oxidase/ROS pathway is essential for the myogenic response of arterioles. Because α1-adrenergic constriction was not associated with nor affected by changes in ROS activity, the role of this pathway may be restricted to mechanotransduction and myogenic constriction.
In cultured, noncontractile VSMCs, oxidant regulation of cell growth is mediated by H2O2.10,11 H2O2 also seems to be the predominant species involved in myogenic constriction, because (1) the myogenic response was associated with increased activity of H2O2, as detected by DCF; (2) the responses were inhibited by catalase but unaffected by a SOD mimic; and (3) exogenous H2O2 was a potent constrictor of arteriolar VSMCs. Studies in large arteries have suggested that oxidant signaling is associated predominantly with diseased phenotypes of VSMCs,10–12 compatible with oxidant regulation of VSMC growth.10,11 Our observations on small arteries are consistent with this proposal and suggest that under physiological conditions, oxidant signaling may be more important in arteriolar compared with arterial VSMCs.
Mouse-tail arterioles represent a novel model of the microcirculation. They demonstrated robust myogenic responses and remarkable spontaneous vasomotion, suggesting that they are a useful and somewhat unique model of the terminal arteriolar vasculature. Several mechanisms have been proposed for the arteriolar myogenic response,1 and there is heterogeneous regulation between different vascular beds.13 The applicability of the present results to other vascular beds is not known. Indeed, although H2O2 can cause constriction through multiple mechanisms,14 it can also act as a vasodilator.15 This suggests that ROS contribution to myogenic constriction may not be universal or indicates a complex regulation of vasomotor responses by ROS.
In conclusion, elevation in PTM causes an NADPH oxidase–dependent generation of ROS in VSMCs of tail arterioles, and these ROS, in particular H2O2, initiate myogenic constriction. Proximal arteries do not participate in this response because of decreased expression of NADPH oxidase, reduced production of H2O2, and decreased ability to constrict to H2O2. Altered regulation of this mechanism may contribute to heightened constriction and oxidant stress in hypertension.5
This work was supported by grants from the Scleroderma Research Foundation and National Institutes of Health (AR46126, HL67331, and HL56091) to N.A.F.
Original received February 16, 2001; revision received June 12, 2001; accepted June 12, 2001.
Davis MJ, Hill MA. Signaling mechanisms underlying the vascular myogenic response. Physiol Rev. 1999; 79: 387–423.
Hall A. Rho GTPases and the actin cytoskeleton. Science. 1998; 279: 509–514.
Wang N, Butler JP, Ingber DE. Mechanotransduction across the cell surface and through the cytoskeleton. Science. 1993; 260: 1124–1127.
Moldovan L, Moldovan NI, Sohn RH, Parikh SA, Goldschmidt-Clermont PJ. Redox changes of cultured endothelial cells and actin dynamics. Circ Res. 2000; 86: 549–557.
Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res. 2000; 86: 494–501.
Suh YA, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D, Chung AB, Griendling KK, Lambeth JD. Cell transformation by the superoxide-generating oxidase Mox1. Nature. 1999; 401: 79–82.
Chotani MA, Flavahan S, Mitra S, Daunt D, Flavahan NA. Silent α2C-adrenergic receptors enable cold-induced vasoconstriction in cutaneous arteries. Am J Physiol. 2000; 278: H1075–H1083.
Jackson SH, Gallin JI, Holland SM. The p47phox mouse knock-out model of chronic granulomatous disease. J Exp Med. 1995; 182: 751–758.
Rubanyi GM, Vanhoutte PM. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am J Physiol. 1986; 250: H822–H827.
Sundaresan M, Yu ZX, Ferrans VJ, Irani K, Finkel T. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science. 1995; 270: 296–299.
Zafari AM, Ushio-Fukai M, Akers M, Yin Q, Shah A, Harrison DG, Taylor WR, Griendling KK. Role of NADH/NADPH oxidase–derived H2O2 in angiotensin II–induced vascular hypertrophy. Hypertension. 1998; 32: 488–495.
Pagano PJ. NAD(P)H oxidase. Arterioscler Thromb Vasc Biol. 2001; 21: 175–177.
Frisbee JC, Roman RJ, Krishna UM, Falck JR, Lombard JH. 20-HETE modulates myogenic response of skeletal muscle resistance arteries from hypertensive Dahl-SS rats. Am J Physiol. 2001; 280: H1066–H1074.
Pelaez NJ, Braun TR, Paul RJ, Meiss RA, Packer CS. H2O2 mediates Ca2+- and MLC20 phosphorylation-independent contraction in intact and permeabilized vascular muscle. Am J Physiol. 2000; 279: H1185–H1193.
Matoba T, Shimokawa H, Nakashima M, Hirakawa Y, Mukai Y, Hirano K, Kanaide H, Takeshita A. Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice. J Clin Invest. 2000; 106: 1521–1530.