This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 92/1/23    most recent 01.RES.0000051860.84509.CEv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Oeckler, R. A. Articles by Wolin, M. S. Search for Related Content PubMed PubMed Citation Articles by Oeckler, R. A. Articles by Wolin, M. S. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB HYDROGEN PEROXIDE Related Collections Peripheral vascular disease Oxidant stress Cell signalling/signal transduction

(Circulation Research. 2003;92:23.)
© 2003 American Heart Association, Inc.

Integrative Physiology

Stretch Enhances Contraction of Bovine Coronary Arteries via an NAD(P)H Oxidase–Mediated Activation of the Extracellular Signal–Regulated Kinase Mitogen-Activated Protein Kinase Cascade

Richard A. Oeckler, Pawel M. Kaminski, Michael S. Wolin

From the Department of Physiology, New York Medical College, Valhalla, NY.

Correspondence to Michael S. Wolin, PhD, Department of Physiology, Basic Science Building, Room 604, New York Medical College, Valhalla, NY 10595. E-mail mike_wolin{at}nymc.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study examines the effects of an increase in passive stretch in endothelium-removed bovine coronary artery on oxidant-induced changes in force generation. Increasing passive stretch on the arterial segments from 5 to 20 g for 20 minutes caused a subsequent increase (P<0.05) in force generation to 30 mmol/L KCl or 0.1 µmol/L serotonin compared with the prestretch control response. Also associated with the passive stretch were increases in superoxide detection by lucigenin and a selective increase in extracellular signal–regulated kinase (ERK) mitogen-activated protein (MAP) kinase phosphorylation measured by Western analysis. The stretch-induced increase in force generation was eliminated by inhibition of the ERK pathway by the MEK inhibitor PD98059 but not by inhibitors of the p38 MAP kinase pathway (SB202190) or c-Jun N-terminal protein kinase pathway (SP200169). Additionally, stretch-induced increases in both ERK phosphorylation and force generation were attenuated by inhibition of tyrosine kinases (genistein), src (PP2), and specific sites on the epidermal growth factor receptor (EGFR) (AG1478). Probes for oxidant signaling, including NAD(P)H oxidase inhibitors (diphenyliodonium and apocynin) or enhancement of peroxide consumption (ebselen) but not inhibition of xanthine oxidase (allopurinol), attenuated the effects of stretch on both ERK phosphorylation and force generation. Furthermore, stretch caused an increase in EGFR phosphorylation and cytosolic to membrane translocation of the p47phox NAD(P)H oxidase subunit. Hydrogen peroxide also elicited contraction through EGFR phosphorylation and ERK. In summary, stretch seems to enhance force generation via ERK signaling through an EGFR/src-dependent mechanism activated by peroxide derived from a stretch-mediated activation of the NAD(P)H oxidase, a response that may contribute to hypertensive alterations in vascular reactivity.

Key Words: stretch • mitogen-activated protein kinases • NAD(P)H oxidase • hydrogen peroxide • oxidant signaling

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
There is recent evidence that mechanical stretch can increase reactive oxygen species (ROS) production¹ and that ROS may be involved with the myogenic response of the microcirculation.² In addition, extracellular signal–regulated kinase (ERK), p38, and c-Jun N-terminal protein kinase (JNK), subsets of the mitogen-activated protein (MAP) kinases, have been shown to be activated by mechanical^3,4 and pulsatile^5–7 stretch as well as oxidants such as peroxide.⁸ Recent studies on the mechanisms of myogenic responses have detected evidence that both the p38 and ERK MAP kinase pathways mediate this response.⁹ However, myogenic responses are not observed in conduit arteries, and evidence for oxidant-induced activation of the MAP kinases has generally been derived from studies in cultured cells, and their subsequent involvement in stretch-induced contractile responses has not yet been established.

Preliminary observations from our laboratory have demonstrated that brief stretching of the coronary vessels increases lucigenin-enhanced chemiluminescence,¹⁰ consistent with previous findings¹ of a stretch-induced increase in superoxide production. Furthermore, these conduit vessels seem to show a novel increased contractile response after exposure to a period of stretch. In preliminary studies using specific inhibitors of the ERK, p38, and JNK MAP kinases, we determined that the ERK MAP kinase signaling appeared necessary for observation of this phenomena and that passive stretch caused a significant increase in ERK, but not p38 or JNK, phosphorylation. Because we found that stretch selectively enhanced ERK phosphorylation and that inhibition of the ERK pathway by the MAP kinase kinase (MEK) inhibitor PD98059 attenuated the stretch-induced enhancement of contractility, we examined whether concurrent increases in superoxide production, MAP kinase activation, and enhanced contractile function were coincidental in nature or instead an interrelated phenomena. In this study we investigated the following: (1) whether oxidant species and their subcellular targets could play a role in modulation of vascular tone; (2) specifically if and how mechanical stretching could activate oxidase activity and superoxide production; (3) the role members of the redox-sensitive ERK MAP kinase cascade might play in the modulation of this response; and (4) the potential role of hydrogen peroxide and epidermal growth factor receptor (EGFR) activation in the signaling mechanism used to activate ERK.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Force Measurement
Isolated endothelium-removed arterial rings were prepared from left anterior descending or circumflex bovine coronary artery (BCA) of calf hearts obtained from a slaughterhouse immediately after slaughter, by adaptation of previously discussed methods.^11,12 In all experiments, the endothelium was removed by gentle rubbing of the lumen of the vessel. Briefly, arterial rings were mounted on wire hooks attached to Grass (FT-03) force displacement transducers for measurement of changes in isometric force on a Grass Instruments polygraph. Arteries were incubated for 1 hour at an optimal passive tension of 5 g in individually thermostated 10-mL tissue baths perfused with Krebs bicarbonate buffer, pH 7.4, at 37°C, gassed with 21% O₂, 5% CO₂, and balance N₂. After 1 hour of equilibration, the vessels were depolarized with Krebs bicarbonate buffer containing KCl in place of NaCl (final concentration, 130 mmol/L KCl). The vessels were then reequilibrated with Krebs bicarbonate for 1 hour before conducting the experiments. The rings were then contracted with 30 mmol/L KCl under 21% O₂ to obtain a control contractile response. In some arteries, stretch-induced force (passive force of 20 g above baseline) was applied to rings for 20 minutes and then returned to basal 5 g of tension. After maximal contraction to a second exposure to 30 mmol/L KCl, the vessels were flash frozen in liquid nitrogen and additionally analyzed via Western blotting. In some studies, arteries were exposed to hydrogen peroxide during the second contraction to 30 mmol/L KCl, or the effects of stretch on the contraction to 0.1 µmol/L serotonin were examined. An experimental tracing of the entire protocol can be seen in Figure 1. Alterations in responses to poststretch contraction to KCl were examined via incubation with the following probes 30 minutes before the period of stretch: 100 µmol/L apocynin (Fluka), 1 µmol/L AG1478, 1 µmol/L PP2 (Cell Signaling Technology), 10 µmol/L genistein, 10 µmol/L PD98059, 50 µmol/L SB202190, 1 µmol/L SP200169, 100 µmol/L ebselen, 10 µmol/L diphenyliodonium (DPI), or 100 µmol/L allopurinol (all from Sigma). Data are reported as the increase in force caused by 30 mmol/L KCl above the 5 g of stretch-induced passive force and normalized to contraction to 30 mmol/L KCl in each artery before exposure to stretch and the probes examined.

View larger version (24K): [in this window] [in a new window]   Figure 1. A, Simultaneous tracings of 10 µmol/L lucigenin-enhanced chemiluminescence for determination of superoxide levels (top) and changes in force (bottom). Note that active contraction to KCl does not alter, whereas a passive stretching and release does alter, superoxide levels in BCA segments. B, Summary data (n=8) of stretch-induced changes in superoxide production as measured by 10 µmol/L lucigenin-enhanced chemiluminescence and the effect of the probes 1 µmol/L PP2, 0.1 mmol/L apocynin, and 10 µmol/L PD98059.

Superoxide Production
Measurements of superoxide levels on exposure to changes in stretch were conducted using the methods used for measurement of force in a single-photon–counting chemiluminescence detection apparatus constructed in a light-tight box.¹¹ In these experiments, coronary artery rings were incubated in Krebs bicarbonate buffer containing 10 µmol/L lucigenin in a continuously gassed cuvette mounted in a thermostated cell holder in front of a photomultiplier tube (Thorn EMI, model 9235B). Photon counting was used to quantitate chemiluminescence. The counts were integrated over 5-second periods, and the analog signal of the integrated counts was continuously recorded on a Grass Model 7 Polygraph together with changes in force.

Western Blotting
Frozen arterial segments were pulverized under liquid nitrogen and placed in a homogenization buffer (60 mmol/L Tris, 10 mmol/L EGTA, 2 mmol/L EDTA, protease and phosphatase inhibitor cocktails [Sigma], pH 7.5). The tubes were spun, the supernatant isolated, and protein levels assayed (Bradford method¹³) for each sample. Ten micrograms of each sample was loaded and run on 12% SDS-PAGE gels, transferred to supported nitrocellulose membranes, and subsequently exposed to primary and secondary antibodies in 5% Milk/TBS-Tween buffers and detected by ECL on autoradiography film. Densitometric analysis was used to quantitate protein levels; phosphorylated enzymes were normalized to total form and then expressed as percent of the control condition. Total and phosphorylated forms of ERK and EGFR antibodies were from Sigma/RBI (St Louis, Mo); p38 and JNK were from Cell Signaling Technology (Beverly, Mass); p47phox antibody was from Santa Cruz (Santa Cruz, Calif); and secondary anti-rabbit and anti-mouse antibodies were from Sigma/RBI.

NAD(P)H Oxidase Subunit Localization
Studies were used via differential centrifugation and subcellular fractionation to identify localization of the NAD(P)H oxidase subunits using Western blotting with commercially available antibodies to p47phox. Briefly, arterial ring segments were freeze clamped either before or in a stretched state after a 20-g passive stretch for 20 minutes. The tissue was then pulverized with mortar and pestle under liquid nitrogen and additionally homogenized in a sucrose-containing buffer (250 mmol/L sucrose, 10 mmol/L HEPES, protease inhibitor cocktail [Sigma], pH 7.4), and the tubes were briefly spun to remove debris. The supernatant was removed and subjected to stepwise centrifugation, first to 10 000g for 10 minutes to isolate mitochondria (discarded) and then to maximum 20 800g. The pellet containing membrane fragments was resuspended in 100 µL of homogenization buffer. Both the resuspended pellet and the supernatant (cytosolic fraction) were additionally analyzed via Western analysis (described above) with the p47phox subunit antibody.

Statistics
Student’s two-tailed t tests were used to assess significance of changes in force generation to 30 mmol/L KCl of the treatments examined compared with the control contraction to KCl. ANOVA with a post hoc Student’s t test using a Bonferroni correction was used to determine significance between experimental groups. Values were represented as mean±SEM, and P<0.05 was used to determine statistical significance.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Stretch-Dependent Alterations in Contractility and the Detection of Superoxide
Preliminary experiments indicated that a brief period of passive stretching of the vessel ring to approximately the maximal level of active force normally generated by these vessel segments causes subsequent increases in superoxide generation and the force developed on exposure to 30 mmol/L KCl. A 20-minute period of stretch was chosen for the study because it caused stable increases in superoxide for longer than a 1-hour period. Time-controlled contractions in unstretched coronary arterial segments developed 5.3±0.6 g of force above 5 g of passive stretch-induced force, compared with 9.5±0.5 g of force in the arteries exposed to stretch or approximately double (186±10%) that seen in prestretched controls. A similar phenomenon was observed to an alternative contractile agent, 0.1 µmol/L serotonin, wherein stretch caused a 178±19% increase in force generation to the same dose of serotonin. As shown by the typical experiment in Figure 1A, concurrent measurement of ROS production revealed a 72% (n=8) increase in the 10 µmol/L lucigenin chemiluminescence-detectable superoxide anion production during the period of stretch. On return of the vessel to the prestretch 5 g of passive force subsequent to the period of stretch, superoxide levels increased an additional 25%, approximately doubling the original prestretch resting levels (see Figure 1). Vessels stretched while being continuously gassed under nitrogen did not demonstrate an enhancement in contractile function (115±21%, n=6), yet, interestingly, on reoxygenation and repeat of the stretch under normal aeration, the stretch response returned (181±25%, n=6), suggesting a role for oxygen in the changes that are observed. Furthermore, Western analysis of stretched coronary arteries (Figure 2) demonstrated a 2-fold increase in the levels of the phosphorylated form of ERK (196±25%, n=12) without increasing the expression of these enzymes; the phosphorylation states of p38 (108±15%, n=8) and JNK (91±21%, n=5) MAP kinases remained unchanged by stretch compared with unstretched control.

View larger version (61K): [in this window] [in a new window]   Figure 2. Representative Western blot analysis of changes in ERK phosphorylation (top) caused by a 20-minute period of passive stretch under 21% O2 versus hypoxia (A) and in the presence and absence of DPI, 10 µmol/L diphenyliodonium (B), and 0.1 mmol/L ebselen (C). D, p38 (left) and JNK (right) phosphorylation remain unchanged by stretch.

Evidence That ROS Mediate the Actions of Stretch
In an effort to elucidate the mechanism involved in the altered contractile reactivity of the vessel induced by stretch and to determine if superoxide levels were indeed central to the mechanism or simply coincidental in nature, we used a variety of probes to analyze changes in force generation (Figure 3A) and ERK phosphorylation (Figure 3B). Treatment with 50 µmol/L allopurinol, a xanthine oxidase (XO) inhibitor, did not prevent the increase in either superoxide production or poststretch contraction. However, an inhibitor of flavoproteins, which include the NAD(P)H oxidase present in BCAs,¹¹ 10 µmol/L DPI, attenuated the contractile response to stretch by an average of 75%. In addition, a more specific inhibitor of the NAD(P)H oxidase, 100 µmol/L apocynin, which prevents the complexing of the cytosolic-to-membrane–bound subunits of the oxidase thought to be necessary for activation,^14,15 also prevented the stretch-induced increase in force generation (Figure 3A) and superoxide (Figure 1B).

View larger version (45K): [in this window] [in a new window]   Figure 3. Influence of reactive oxygen species intermediates in the response to stretch. Changes in force generation (A) and ERK phosphorylation (B) in BCA ring segments, either incubated (time control) or passively stretched for 20 minutes, in the presence of the following probes: 10 µmol/L DPI, 0.1 mmol/L apocynin, 50 µmol/L allopurinol, or the glutathione peroxidase mimetic 0.1 mmol/L ebselen (n=8).

Changes in ERK phosphorylation caused by stretch were measured in the same animals by examining ring segments from the force generation experiments frozen at the end of the protocol in a contracted state. The stretch-induced doubling of ERK phosphorylation was inhibited by stretching the vessel segments while under hypoxia (Figure 2A). Additionally, the increase in phosphorylation was reduced an average of 85% by DPI and 64% by apocynin, whereas the xanthine oxidase inhibitor allopurinol did not prevent the increase in ERK phosphorylation caused by stretch (Figure 3B). Hence, the ERK phosphorylation changes seem to parallel the changes in force generation (Figure 3A versus Figure 3B), suggesting a possible mechanistic link between the two through comparison of the summary data (online Tables 1 and 2, available in the data supplement at http://www.circresaha.org).

To additionally differentiate between possible oxidant mediators of the stretch phenomenon, the glutathione peroxidase mimetic, 0.1 mmol/L ebselen,¹² was used to scavenge peroxide formed via action of cellular oxidases and superoxide dismutases. Vessels treated with ebselen and then stretched showed no significant difference from unstretched vessels; the stretch-enhanced increase in force generation was inhibited by 83%. Ebselen also attenuated the stretch-induced increase in ERK phosphorylation by 65% (Figure 3). These data implicate superoxide-derived hydrogen peroxide as a participant in the stretch-induced enhancement in contractility seen in these arterial segments.

ERK MAP Kinase Inhibition Attenuates the Actions of Stretch on Force Generation
Recent evidence suggests that oxidants may activate the ERK family of MAP kinases, which have been shown to promote contraction, possibly through the phosphorylation of caldesmon or calponin, two mediators of the contractile apparatus.^16–18 To determine if this pathway was involved in our model, changes in ERK phosphorylation were measured by Western analysis in parallel with changes in force. The activation of MEK was inhibited by 10 µmol/L PD98059, a probe that specifically and reversibly prevents phosphorylation and subsequent activation of ERK.¹⁹ As indicated by the summary data in Figure 4, the MEK inhibitor attenuated the stretch-induced increase in force generation without altering the control response to either KCl or serotonin. For example, the MEK inhibitor reduced the expression of this increase in force attributable to stretch by up to 85%, whereas the contraction to 30 mmol/L KCl or 0.1 µmol/L serotonin was not altered by the presence of the MEK inhibitor in unstretched time-control vessels. The presence of the MEK inhibitor PD98059 also reduced the phosphorylation of ERK to 64±19%, n=8, in the stretched vessels and decreased the levels of phosphorylation by 82±15%, n=8, in the control arteries. Similar experiments were conducted with inhibitors of the p38 (50 µmol/L SB202190²⁰) and JNK (1 µmol/L SP200169) MAP kinase pathways. Neither SB202190 nor SP200169 significantly attenuated either the increase in contractile response or ERK phosphorylation caused by stretch, and neither inhibitor significantly altered ERK phosphorylation levels (see Figure 4). Additionally, neither the pharmacologic probes nor the vehicle 0.1% DMSO altered contractile responses in arteries that were not exposed to stretch (see online Table 2).

View larger version (50K): [in this window] [in a new window]   Figure 4. Influence of kinase signaling pathways in response to stretch. Force generation (A) and ERK phosphorylation (B) summary data (n=8) of BCA ring segments, either incubated (time control) or passively stretched for 20 minutes, in the presence of the following probes: 1 µmol/L genistein, 10 µmol/L PD98059, 50 µmol/L SB202190, 1 µmol/L SP200169, 1 µmol/L PP2, or 1 µmol/L AG1478.

Involvement of EGFR and src in Stretch-Induced Signaling
We next examined upstream of ERK in an attempt to dissect the pathway activated by the initial peroxide-protein interaction. The tyrosine kinase inhibitor 1 µmol/L genistein prevented both the increase in force generation (69±15%, n=6) and ERK phosphorylation (65±35%) caused by stretch, thus suggesting a role for protein tyrosine kinases in our model. The EGFR and its associated signaling, including src, a tyrosine kinase related to EGFR signaling pathways, have recently been implicated as being potentially redox sensitive²¹ and involved in stretch-induced ERK activation.⁶ Western analysis using antibodies to tyrosine residues of the EGFR known to lead to ERK pathway activation (Tyr 1068)²¹ indicates that stretch causes a 174±36% increase in phosphorylation over control conditions (Figure 5). Furthermore, a specific inhibitor of src, PP2,²² and an inhibitor of EGFR tyrosine kinase, AG1478,²³ prevented both the stretch-induced increases in EGFR phosphorylation and downstream ERK phosphorylation (115±18%) and the enhancement in the contractile response (96±22%) caused by stretch (Figures 4 and 5B). Because src has been reported to promote a prolonged activation of NAD(P)H oxidase,²⁴ the PP2 inhibitor was used to examine whether src contributed to stretch-induced oxidase activation. As shown in Figure 1B, the increase in superoxide derived by stretch was not altered by src inhibition with PP2.

View larger version (31K): [in this window] [in a new window]   Figure 5. A, Representative blot from Western analysis of phosphorylation changes at tyrosine residue 1068 of the EGFR in control and stretched BCA segments. Results were normalized to antibodies against nonphosphorylated EGFR and also to actin (shown). B, Summary data demonstrating that 1 µmol/L PP2, 0.1 mmol/L apocynin, and 10 µmol/L AG1478 prevent the stretch-induced phosphorylation of tyrosine residue 1068 of the EGFR (n=8).

Hydrogen Peroxide Elicits Contraction Through EGFR and ERK
The effects of the AG1478 EGFR inhibitor and the PD98059 ERK inhibitor on responses of arteries precontracted with 30 mmol/L KCl to 0.1 mmol/L and 1 mmol/L hydrogen peroxide were examined. As shown in Figure 6A, the modest relaxation to 0.1 mmol/L was enhanced and the contraction to 1 mmol/L peroxide was converted to a relaxation by the EGFR and ERK inhibitors. Peroxide (1 mmol/L) was also observed to cause an increase in EGFR phosphorylation at Tyr 1068, which was inhibited by AG1478 (Figure 6B), and increase ERK phosphorylation, which was attenuated by both AG1478 and PD98059 (Figure 6C).

View larger version (20K): [in this window] [in a new window]   Figure 6. A, Summary data of the changes in force caused by either 0.1 mmol/L (left) or 1 mmol/L (right) hydrogen peroxide and the effects of 10 µmol/L PD98059 (gray bars) and 1 µmol/L AG1478 (black bars) on these responses. Inhibition of ERK and EGFR signaling enhance relaxation to peroxide at the lower dose and convert contractile to relaxation responses at the higher (1 mmol/L) level of peroxide (n=8). B, Summary data demonstrating the increase in the phosphorylation of tyrosine residue 1068 of the EGF receptor by 1 mmol/L hydrogen peroxide and the attenuation of the response in the presence of 10 µmol/L AG1478 (n=6). C, Summary data of the increase in ERK phosphorylation caused by 1 mmol/L peroxide and the prevention of peroxide’s effects in the presence of 10 µmol/L PD98059 (n=8) and 1 µmol/L AG1478.

Stretch-Induced Translocation of Oxidase Subunits
The NAD(P)H oxidase is composed of cytosolic and membrane-bound subunits that complex to form the active, superoxide-generating unit.^15,25 We used antibodies to the cytosolic p47phox subunit of the oxidase and differential centrifugation techniques to determine whether stretch caused a translocation of this subunit to the membrane. Figure 7 demonstrates an increase in p47phox levels in the membrane fraction of stretched tissues versus unstretched controls. In stretched vessels, p47phox levels increased to 154±40% of control in the membrane and decreased to 62±22% of control in the cytosolic fraction, suggesting that a translocation is occurring under stretch. Furthermore, the presence of 100 µmol/L apocynin attenuated the apparent membrane translocation of p47phox (summary data in Figure 7B).

View larger version (46K): [in this window] [in a new window]   Figure 7. A, Representative Western analysis of p47phox subunits in both the membrane (top) and cytosolic (bottom) fractions of control and stretched BCA segments that were obtained via differential centrifugation of BCA ring segments. B, Summary data showing the prevention by 0.l mmol/L apocynin of the stretch-induced increase in translocation of the p47phox subunit from the cytosol to membrane fractions (n=6).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we found evidence for a stretch-induced, oxidant-dependent enhancement of contractile force via activation of the NAD(P)H oxidase and a subset of the MAP kinase signaling pathway. Stretch caused a selective increase in ERK, but not p38 or JNK, phosphorylation without increasing its expression, and inhibition of ERK by the selective MEK inhibitor PD98059 attenuated the increase in both phosphorylation and force generation, whereas p38 and JNK inhibition did not. Furthermore, both the changes in force development and ERK activation seem to involve a ROS-dependent activation of the EGFR, because probes that targeted the NAD(P)H oxidase, scavenged superoxide-derived peroxide, or inhibited EGFR phosphorylation linked to ERK activation all prevented the stretch-induced enhancement in both force development and ERK phosphorylation.

Stretch-Induced Oxidant Production and the Role of NAD(P)H Oxidase
It has been shown previously that increased transmural pressure can cause an increased production of superoxide in the vessel wall.²⁶ Additionally, mechanisms have been suggested for the role of oxidant species in both myogenic tone and in the mediation of wall shear stress mechanotransduction pathways. A recent study of the myogenic response in rat arteries and arterioles found the response to be abrogated by either the scavenging of peroxide or inhibition of NAD(P)H oxidase.² The present study demonstrates that stretch, an additional physiologically relevant force in vivo, has a ROS component in large conduit arteries as well. The prolonged increase in superoxide production remained elevated for more than 1 hour after the cessation of stretch, suggesting an activation of an oxidase source being caused by the physical force.

The two likely potential sources of this stretch-induced increase in superoxide levels are the flavin-containing proteins XO and NAD(P)H oxidase. In this study, we demonstrate that poststretch increases in force generation are attenuated by the scavenging of peroxide via the glutathione peroxidase mimetic ebselen, the NAD(P)H oxidase-specific probe apocynin, and the flavoprotein-inhibitor diphenyliodonium but not by the inhibition of XO. These data are consistent with previous observations from our group¹¹ detecting high DPI-inhibitable NADH oxidase activity and low XO activity in BCA, suggesting that an NAD(P)H oxidase could potentially be the source of stretch-induced superoxide production. Paralleling changes in force generation, the stretch-induced increase in ERK phosphorylation was also inhibited by DPI, apocynin, and ebselen but not allopurinol. Hence, there seems to be a prominent role for ROS in the stretch response, in particular a superoxide-derived hydrogen peroxide activation of the ERK MAP kinase cascade. Because these probes did not inhibit basal levels of ERK phosphorylation in unstretched arteries contracted with KCl or serotonin, mechanisms involving ROS do not seem to contribute to the basal activation of ERK under these conditions.

As previously mentioned, the nonphagocytic NAD(P)H oxidase is comprised of both cytosolic and membrane-bound components. Combinations of the cytosolic p47phox, p67phox, and rac-1 associate, translocate to, bind, and activate the membrane-bound complex composed of the p22phox and gp91phox (or nox homologue) subunits.²⁵ Cytosolic subunit binding is thought to reduce molecular oxygen to superoxide. Apocynin has been shown to prevent the binding of the cytosolic subunits of the NAD(P)H oxidase to the membrane-bound p22phox/gp91phox subunits, preventing oxidase activation and subsequent production of superoxide.^14,15 Additionally implicating the NAD(P)H oxidase in the response to stretch, incubation of vessels with apocynin prevented the stretch-induced increase in force generation, superoxide detection, and ERK phosphorylation while having no statistically significant effect on either component in the unstretched controls segments.

Oxidase Activation by Stretch
To determine the causal role for stretch in oxidase activation, the translocation of the cytosolic p47phox subunit to the membrane oxidase subunits was targeted for Western analysis. Arterial segments that were either exposed to a 20-minute passive stretch or not were then processed additionally by differential centrifugation for the isolation of cytosolic and membrane fractions and compared for relative change in p47phox subunit levels by Western analysis (Figure 7). Because of basal activity of the oxidase, it was assumed that some p47phox might be present in the membrane fraction under resting, unstretched conditions but that increases in overall p47phox levels would be caused by stretch. Indeed, the amount of p47phox found in the membrane fraction increased by >50% of control unstretched levels because of stretch, while decreasing in the cytosolic fraction by a similar amount (46%). This suggests that a translocation of the p47phox subunit is a result of the mechanism of oxidase activation by stretch. As expected, we did find p47phox protein in control unstretched arterial membrane fractions, and this potentially contributes to the observed low basal activity of the oxidase. Interpretation of these data allows us to begin to implicate a specific mechanism of activation of the NAD(P)H oxidase coupled to a subsequent ROS-mediated signaling in the contractile changes caused by physical stretch.

Stretch-Induced Protein Tyrosine Kinase and ERK MAP Kinase Signaling
The involvement of ROS in multiple cellular signaling processes has been established and discussed previously in review articles.^27,28 In cultured smooth muscle cells, ERK phosphorylation through activation of the EGFR has been shown to be stimulated by mechanical stretch,^5–7 and in vivo balloon catheterization stretch of porcine arteries also promotes ERK phosphorylation.⁴ Evidence for ROS-dependent linkage of physical forces to MAP kinase activation has been demonstrated in pulsatile-stretch experiments in vitro⁶ as well as to receptor mechanisms in cell culture studies; however, these studies have focused on their role in the apoptotic and hypertrophic growth pathways involving the MAP kinases^4,6 instead of a possible contractile function. In this study, we demonstrate that stretch of BCAs causes a selective activation of the ERK MAP kinase pathway and that the subsequent expression of enhanced force generation is attenuated by selective inhibition of MEK. Although others have shown p38 or JNK to be affected by stretch^29–31 and possibly modulate smooth muscle contraction via ERK phosphorylation of caldesmon,³¹ our results show no evidence for either changes in p38 or JNK phosphorylation by stretch or changes in force generation after inhibition in our model. The inability of the ERK inhibitor to alter contraction in unstretched KCl or serotonin control vessels may have a 2-fold importance. First, PD98059 in BCA does not demonstrate nonspecific effects on K⁺ channels as has recently been suggested.³² Second, the basal activation of these pathways does not seem to contribute to resting force generation, but instead ERK signaling may be of increased importance under pathological conditions, such as increased pressures.

The initial peroxide-protein interaction remains poorly understood. Superoxide and peroxide have been implicated in the activation of tyrosine kinases and inhibition of phosphatases both by direct interaction³³ as well as indirectly via ROS signaling from upstream mediators such as focal adhesion kinase³⁴ and angiotensin II type I receptor activation.²⁴ The findings presented here support roles for src activation and EGF receptor phosphorylation signaling in the ERK-mediated response to stretch. Exogenous hydrogen peroxide was observed to activate contraction through the EGFR-ERK pathway and to stimulate a previously studied relaxation response that seems to be mediated through cGMP.^11,12,35 We are presently unable to exclude the possibility of additional signaling components, including phosphatases upstream of src or EGFR, as the site of action of peroxides. However, the absence of detection of a relaxation response to stretch after inhibition of ERK suggests that the NAD(P)H oxidase involved may be located in the proximity of src and EGFR.

Influence of Stretch-Induced ERK Signaling on Vascular Function
This study seems to provide novel functional evidence for the model shown in Figure 8, linking stretch to a prolonged activation of NAD(P)H oxidase and a peroxide-mediated stimulation of ERK MAP kinase through a src-EGFR signaling pathway and enhancing arterial contractile responses in BCAs. Although the mechanism promoting p47phox subunit translocation leading NAD(P)H oxidase production of superoxide anion remains unclear, recent evidence suggests that integrin and subcellular structural proteins such as actin fibers may be involved in cellular mechanotransduction pathways.³⁶ Recent findings also suggest that ERK signaling may ultimately alter contractile function through changes in Ca²⁺ or modulation of contractile regulatory proteins, including caldesmon and calponin, leading to altered actin-myosin crossbridge interaction.^16–18 In arterioles, wall stress seems to activate both force generation and growth-mediated responses through src-dependent ERK-mediated mechanisms, and the resulting myogenic tone is thought to reduce wall stress and expression of the remodeling response.³⁷ The stretch-induced enhancement of reactivity to contractile agents observed in the present study could be of importance in enhancing the reactivity of conduit arteries and controlling remodeling responses under hypertensive conditions.

View larger version (36K): [in this window] [in a new window]   Figure 8. Model of the proposed mechanism by which stretch alters contractile function in BCA through activation of NAD(P)H oxidase and the ERK MAP kinase pathway. PD98059 indicates MEK inhibitor; SB201290, p38 inhibitor; SP200169, JNK inhibitor; PP2, src-specific tyrosine kinase inhibitor; and AG1478, EGFR tyrosine kinase inhibitor.

	Acknowledgments

 
This work was supported by United States Public Health Service grants HL31069, HL43023, and HL66331. We thank Mary Elizabeth Arcuino for providing technical assistance with the Western analysis studies.

Received December 18, 2001; revision received November 27, 2002; accepted November 27, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Howard B, Alexander RW, Nerem RM, Griendling KK, Taylor WR. Cyclic strain induces an oxidative stress in endothelial cells. Am J Physiol Cell Physiol. 1997; 272: C421–C427.[Abstract/Free Full Text]
2. Nowicki PT, Flavahan S, Hassanain H, Mitra S, Holland S, Goldschmidt-Clermont PJ, Flavahan NA. Redox signaling of the arteriolar myogenic response. Circ Res. 2001; 89: 114–116.[Abstract/Free Full Text]
3. Komuro I, Kudo S, Yamazaki T, Zou Y, Shiojima I, Yazaki Y. Mechanical stretch activates the stress-activated protein kinases in cardiac myocytes. FASEB J. 1996; 10: 631–636.[Abstract]
4. Pyles JM, March KL, Franklin M, Mehdi K, Wilensky RL, Adam LP. Activation of MAP kinase in vivo follows balloon overstretch injury of porcine coronary and carotid arteries. Circ Res. 1997; 81: 904–910.[Abstract/Free Full Text]
5. Seko Y, Takahashi N, Tobe K, Kadowaki T, Yazaki Y. Pulsatile stretch activates mitogen-activated protein kinase (MAPK) family members and focal adhesion kinase (p125^FAK) in cultured rat cardiac myocytes. Biochem Biophys Res Commun. 1999; 259: 8–14.[CrossRef][Medline] [Order article via Infotrieve]
6. Iwasaki H, Eguchi S, Ueno H, Marumo F, Hirata Y. Mechanical stretch stimulates growth of vascular smooth muscle cells via epidermal growth factor receptor. Am J Physiol Heart Circ Physiol. 2000; 278: H521–H529.[Abstract/Free Full Text]
7. Lehoux S, Esposito B, Merval R, Loufrani L, Tedgui A. Pulsatile stretch-induced extracellular signal-regulated kinase 1/2 activation in organ culture of rabbit aorta involves reactive oxygen species. Arterioscler Thromb Vasc Biol. 2000; 20: 2366–2372.[Abstract/Free Full Text]
8. 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.[Abstract/Free Full Text]
9. Wu Z, Woodring PJ, Bhakta KS, Tamura K, Wen F, Feramisco JR, Karin M, Wang JY, Puri PL. p38 and extracellular signal-regulated kinases regulate the myogenic program at multiple steps. Mol Cell Biol. 2000; 20: 3951–3964.[Abstract/Free Full Text]
10. Oeckler RA, Kaminski PM, Wolin MS. Pretreatment of coronary arteries with elevated force results in increased contractile reactivity through activation of the p42/p44 MAP kinase cascade. Circulation. 2000; 102 (suppl II): II–14.Abstract.
11. Mohazzab-H KM, Kaminski PM, Fayngersh RP, Wolin MS. Oxygen-elicited responses in calf coronary arteries: role of H₂O₂ production via NADH-derived superoxide. Am J Physiol. 1996; 270: H1044–H1053.[Medline] [Order article via Infotrieve]
12. Mohazzab-H KM, Agarwal R, Wolin MS. Influence of glutathione peroxidase on coronary artery responses to alterations in PO₂ and H₂O₂. Am J Physiol. 1999; 276: H235–H241.[Medline] [Order article via Infotrieve]
13. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72: 248–254.[CrossRef][Medline] [Order article via Infotrieve]
14. Stolk J, Hiltermann TJ, Dijkman JH, Verhoeven AJ. Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol. Am J Respir Cell Mol Biol. 1994; 11: 95–102.[Abstract]
15. Babior BM. NADPH oxidase: an update. Blood. 1999; 93: 1464–1476.[Free Full Text]
16. Menice CB, Hulvershorn J, Adam LP, Wang CA, Morgan KG. Calponin and mitogen-activated protein kinase signaling in differentiated vascular smooth muscle. J Biol Chem. 1997; 272: 25157–25161.[Abstract/Free Full Text]
17. Leinweber BD, Leavis PC, Grabarek Z, Wang CL, Morgan KG. Extracellular regulated kinase (ERK) interaction with actin and the calponin homology (CH) domain of actin-binding proteins. Biochem J. 1999; 344(pt 1): 117–123.[Medline] [Order article via Infotrieve]
18. Lee YH, Gallant C, Guo H, Li Y, Wang CA, Morgan KG. Regulation of vascular smooth muscle tone by N-terminal region of caldesmon: possible role of tethering actin to myosin. J Biol Chem. 2000; 275: 3213–3220.[Abstract/Free Full Text]
19. Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A. 1995; 92: 7686–7689.[Abstract/Free Full Text]
20. Nemoto S, Xiang J, Huang S, Lin A. Induction of apoptosis by SB202190 through inhibition of p38ß mitogen-activated protein kinase. J Biol Chem. 1998; 273: 16415–16420.[Abstract/Free Full Text]
21. Zwick E, Hackel PO, Prenzel N, Ullrich A. The EGF receptor as central transducer of heterologous signalling systems. Trends Pharmacol Sci. 1999; 20: 408–412.[CrossRef][Medline] [Order article via Infotrieve]
22. Salazar EP, Rozengurt E. Bombesin and platelet-derived growth factor induce association of endogenous focal adhesion kinase with Src in intact Swiss 3T3 cells. J Biol Chem. 1999; 274: 28371–28378.[Abstract/Free Full Text]
23. Osherov N, Levitzki A. Epidermal growth factor dependent activation of Src family kinases. Eur J Biochem. 1994; 225: 1047–1053.[Medline] [Order article via Infotrieve]
24. Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, Griendling KK. Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators. Circ Res. 2002; 91: 406–413.[Abstract/Free Full Text]
25. Lassègue B, Sorescu D, Szöcs K, Yin Q, Akers M, Zhang Y, Grant SL, Lambeth JD, Griendling KK. Novel gp91^phox homologues in vascular smooth muscle cells: nox1 mediates angiotensin II–induced superoxide formation and redox-sensitive signaling pathways. Circ Res. 2001; 88: 888–894.[Abstract/Free Full Text]
26. Wei EP, Kontos HA, Christman CW, DeWitt DS, Povlishock JT. Superoxide generation and reversal of acetylcholine-induced cerebral arteriolar dilation after acute hypertension. Circ Res. 1985; 57: 781–787.[Abstract/Free Full Text]
27. Wolin MS. Interactions of oxidants with vascular signaling systems. Arterioscler Thromb Vasc Biol. 2000; 20: 1430–1442.[Abstract/Free Full Text]
28. Griendling KK, Sorescu D, Lassegue B, Ushio-Fukai M. Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arterioscler Thromb Vasc Biol. 2000; 20: 2175–2183.[Abstract/Free Full Text]
29. Nguyen HT, Adam RM, Bride SH, Park JM, Peters CA, Freeman MR. Cyclic stretch activates p38 SAPK2-, ErbB2-, and AT1-dependent signaling in bladder smooth muscle cells. Am J Physiol Cell Physiol. 2000; 279: C1155–C1167.[Abstract/Free Full Text]
30. Sawada Y, Nakamura K, Doi K, Takeda K, Tobiume K, Saitoh M, Morita K, Komuro I, De Vos K, Sheetz M, Ichijo H. Rap1 is involved in cell stretching modulation of p38 but not ERK or JNK MAP kinase. J Cell Sci. 2001; 114: 1221–1227.[Abstract]
31. Yamboliev IA, Hedges JC, Mutnick JL, Adam LP, Gerthoffer WT. Evidence for modulation of smooth muscle force by the p38 MAP kinase/HSP27 pathway. Am J Physiol Heart Circ Physiol. 2000; 278: H1899–H1907.[Abstract/Free Full Text]
32. Lagaud GJ, Lam E, Lui A, van Breemen C, Laher I. Nonspecific inhibition of myogenic tone by PD98059, a MEK1 inhibitor, in rat middle cerebral arteries. Biochem Biophys Res Commun. 1999; 257: 523–527.[CrossRef][Medline] [Order article via Infotrieve]
33. Wolin MS, Gupte SA, Oeckler RA. Superoxide in the vascular system. J Vasc Res. 2002; 39: 1–16.[CrossRef][Medline] [Order article via Infotrieve]
34. Wang JG, Miyazu M, Matsushita E, Sokabe M, Naruse K. Uniaxial cyclic stretch induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation. Biochem Biophys Res Commun. 2001; 288: 356–361.[CrossRef][Medline] [Order article via Infotrieve]
35. Burke TM, Wolin MS. Hydrogen peroxide elicits pulmonary arterial relaxation and guanylate cyclase activation. Am J Physiol. 1987; 252: H721–H732.[Medline] [Order article via Infotrieve]
36. Numaguchi K, Eguchi S, Yamakawa T, Motley ED, Inagami T. Mechanotransduction of rat aortic vascular smooth muscle cells requires RhoA and intact actin filaments. Circ Res. 1999; 85: 5–11.[Abstract/Free Full Text]
37. Prewitt RL, Rice DC, Dobrian AD. Adaptation of resistance arteries to increases in pressure. Microcirculation. 2002; 9: 295–304.[CrossRef][Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				R. A. Oeckler and R. D. Hubmayr Ventilator-associated lung injury: a search for better therapeutic targets Eur. Respir. J., December 1, 2007; 30(6): 1216 - 1226. [Abstract] [Full Text] [PDF]

				T. Nagaoka, T. W. Hein, A. Yoshida, and L. Kuo Resveratrol, a Component of Red Wine, Elicits Dilation of Isolated Porcine Retinal Arterioles: Role of Nitric Oxide and Potassium Channels Invest. Ophthalmol. Vis. Sci., September 1, 2007; 48(9): 4232 - 4239. [Abstract] [Full Text] [PDF]

				A. Jacobson, C. Yan, Q. Gao, T. Rincon-Skinner, A. Rivera, J. Edwards, A. Huang, G. Kaley, and D. Sun Aging enhances pressure-induced arterial superoxide formation Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1344 - H1350. [Abstract] [Full Text] [PDF]

				M. J. Haurani and P. J. Pagano Adventitial fibroblast reactive oxygen species as autacrine and paracrine mediators of remodeling: Bellwether for vascular disease? Cardiovasc Res, September 1, 2007; 75(4): 679 - 689. [Abstract] [Full Text] [PDF]

				M. N. Richard, J. F. Deniset, A. L. Kneesh, D. Blackwood, and G. N. Pierce Mechanical Stretching Stimulates Smooth Muscle Cell Growth, Nuclear Protein Import, and Nuclear Pore Expression through Mitogen-activated Protein Kinase Activation J. Biol. Chem., August 10, 2007; 282(32): 23081 - 23088. [Abstract] [Full Text] [PDF]

				L. Ding, A. Chapman, R. Boyd, and H. D. Wang ERK activation contributes to regulation of spontaneous contractile tone via superoxide anion in isolated rat aorta of angiotensin II-induced hypertension Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H2997 - H3005. [Abstract] [Full Text] [PDF]

				L. S. Chaturvedi, H. M. Marsh, and M. D. Basson Src and focal adhesion kinase mediate mechanical strain-induced proliferation and ERK1/2 phosphorylation in human H441 pulmonary epithelial cells Am J Physiol Cell Physiol, May 1, 2007; 292(5): C1701 - C1713. [Abstract] [Full Text] [PDF]

				L. O. Lerman and A. Lerman All Oxidase Roads Lead to Angiotensin, Too Arterioscler. Thromb. Vasc. Biol., April 1, 2007; 27(4): 703 - 704. [Full Text] [PDF]

				S. Papaiahgari, A. Yerrapureddy, P. M. Hassoun, J. G. N. Garcia, K. G. Birukov, and S. P. Reddy EGFR-Activated Signaling and Actin Remodeling Regulate Cyclic Stretch-Induced NRF2-ARE Activation Am. J. Respir. Cell Mol. Biol., March 1, 2007; 36(3): 304 - 312. [Abstract] [Full Text] [PDF]

				F. Shi, Y.-J. Chiu, Y. Cho, T. A. Bullard, M. Sokabe, and K. Fujiwara Down-regulation of ERK but not MEK phosphorylation in cultured endothelial cells by repeated changes in cyclic stretch Cardiovasc Res, March 1, 2007; 73(4): 813 - 822. [Abstract] [Full Text] [PDF]

				P. Cong, Z.-L. Xiao, P. Biancani, and J. Behar Prostaglandins mediate tonic contraction of the guinea pig and human gallbladder Am J Physiol Gastrointest Liver Physiol, January 1, 2007; 292(1): G409 - G418. [Abstract] [Full Text] [PDF]

				F. M. Kouri and O. Eickelberg Transforming Growth Factor-{alpha}, a Novel Mediator of Strain-Induced Vascular Remodeling Circ. Res., August 18, 2006; 99(4): 348 - 350. [Full Text] [PDF]

				C. A. Lemarie, P.-L. Tharaux, B. Esposito, A. Tedgui, and S. Lehoux Transforming Growth Factor-{alpha} Mediates Nuclear Factor {kappa}B Activation in Strained Arteries Circ. Res., August 18, 2006; 99(4): 434 - 441. [Abstract] [Full Text] [PDF]

				T. M. Paravicini and R. M. Touyz Redox signaling in hypertension Cardiovasc Res, July 15, 2006; 71(2): 247 - 258. [Abstract] [Full Text] [PDF]