Cytosolic NADH redox and thiol oxidation regulate pulmonary arterial force through ERK MAP kinase

Richard A. Oeckler,1 Elizabeth Arcuino,1 Mansoor Ahmad,1 Susan C. Olson,2 and Michael S. Wolin1

Departments of 1Physiology and 1Biochemistry and 2Molecular Biology, New York Medical College, Valhalla, New York

Submitted 11 June 2004 ; accepted in final form 15 January 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An ERK MAP kinase-mediated contractile mechanism previously reported to be activated by peroxide and stretch in bovine coronary arteries is shown in this study to be present in endothelium-denuded bovine pulmonary arteries and subject to regulation by modulation of cytosolic NAD(H) redox through the lactate dehydrogenase reaction. Although our previous work identified an acute PO2-dependent peroxide-mediated relaxation of bovine pulmonary arteries on exposure to lactate, a 30-min treatment with 10 mM lactate enhanced ERK phosphorylation and increased force generation to 30 mM KCl. Hypoxia inhibited these responses to lactate. Increases in ERK phosphorylation and the enhancement of force generation by lactate and stretch are attenuated in the presence of inhibitors of Nox oxidase (0.1 mM apocynin) or ERK activation (10 µM PD-98059) and by 0.1 mM ebselen. Additionally, incubation of pulmonary arteries with 10 mM pyruvate lowered basal levels of ERK phosphorylation, and it inhibited both the ERK phosphorylation and the enhancement in force generation to 30 mM KCl caused by stretch. Treatment of pulmonary arteries with the thiol oxidant diamide (1 µM) elicited what appears to be a peroxide-independent increase in force and ERK phosphorylation that were both attenuated by PD-98059. Thus pulmonary arteries possess a peroxide-elicited contractile mechanism involving ERK MAP kinase, which is stimulated by stretch and regulated through the control of Nox oxidase activity by the availability of cytosolic NADH.

diamide; lactate; Nox; peroxide; stretch


OUR PREVIOUS STUDIES HAVE provided evidence that lactate can cause relaxation of endothelium-denuded bovine pulmonary arteries and several other vascular segments through a cGMP mechanism that appears to involve hydrogen peroxide (H2O2) derived from increasing the level of cytosolic NADH and the activity of a superoxide-generating Nox-type oxidase (4, 7, 18, 22). When Cu,Zn-superoxide-dismutase activity is inhibited, relaxation of bovine pulmonary arteries to nitric oxide is attenuated by increases in superoxide in a manner that is enhanced by lactate and decreased by pyruvate, suggesting that the lactate dehydrogenase (LDH) reaction can control superoxide production through its influence on cytosolic NAD(H) redox (9). In our recent studies in endothelium-denuded bovine coronary arteries, we detected evidence for the presence of an additional contractile mechanism to peroxide mediated through an src-EGF receptor-dependent activation of ERK MAP kinase (21). In these studies, an initial treatment with passive stretch was observed to increase the contractile response to KCl and serotonin in a manner that appeared to be dependent on peroxide derived from a stretch-mediated activation of superoxide generation by a Nox-type oxidase, which was inhibited by apocynin and diphenyliodonium (DPI). Thus the LDH reaction appears to be able to control the generation of vasoactive levels of superoxide and H2O2 in vascular smooth muscle through its influence on cytosolic NAD(H) redox, and H2O2 has the ability to regulate processes that promote both relaxation and contraction.

In this study, we examine whether bovine pulmonary arteries show a peroxide-mediated contractile response that functions through the ERK MAP kinase pathway in addition to the previously studied cGMP-associated relaxation response (3) and whether a passive stretch treatment enhances contractile force through activation of this pathway. Then the actions of pyruvate and lactate are examined to determine whether the hypothesized effects of these metabolites on cytosolic NAD(H) redox regulate basal and stretch-enhanced force generation through changes in the ERK MAP kinase pathway. While studying the vasoactive actions of the thiol oxidant diamide, we observed this agent also caused a direct contractile response correlated with activation of the ERK MAP kinase system, and we examined this response to determine whether it functioned through modulating the effects of endogenous H2O2.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Force measurement. Isolated endothelium-denuded arterial rings of 3–4 mm in length and diameter were prepared from secondary branches of the bovine pulmonary arteries of calf lungs obtained from a slaughterhouse immediately after slaughter, by adaptation of previously discussed methods (3, 21, 22). In all experiments, the endothelium was denuded by gentle rubbing of the lumen of the vessel. In brief, arterial rings were mounted on wire hooks attached to Coulbourne force displacement transducers for measurement of changes in isometric force via an AD Instrument's Powerlab system running Chart 5 software. Arteries were incubated for 1 h at an optimal passive tension of 5 g in individually thermostated 10-ml tissue baths containing Krebs-bicarbonate buffer, pH 7.4, containing (in mM): 118 NaCl, 4.7 KCl, 1.5 CaCl2, 25 NaHCO3, 1.1 MgSO4, 1.2 KH2PO4, and 5.6 glucose, at 37°C gassed with 21% O2, 5% CO2, balance N2. After 1 h of equilibration, the vessels were depolarized with Krebs-bicarbonate buffer containing KCl in place of NaCl (final concentration 130 mM KCl). The vessels were then re-equilibrated with Krebs-bicarbonate for 1 h. The rings were then contracted with 30 mM KCl (prepared by substituting NaCl with KCl, "Pre" in Figs. 3A and 4A) under 21% O2, to generate a control contraction. In some arteries, stretch was used to increase the passive force to 20 g by lengthening the arterial rings with the rack and pinion manipulator used to adjust the position of the force transducer for 20 min. Force was readjusted to compensate for stretch-induced decays in force, as needed, and then passive force was returned to the basal level (5 g) before the second exposure to 30 mM KCl ("Post" in Fig. 3A). After force generation elicited by a second exposure to 30 mM KCl in the absence or presence of drugs indicated in the results reached a steady state, the vessels were rapidly frozen in liquid nitrogen and analyzed further via Western blotting. In some studies, pretreatment with 10 mM lactate or 10 mM pyruvate was used to examine redox modulation, and an atmosphere of 95% N2-5% CO2 was used for probing the effects of hypoxia. As described previously (21), 100 µM apocynin (Fluka, St. Louis, MO), 100 µM ebselen, and 10 µM PD-98059 (Sigma, St. Louis, MO) were used to examine the influence of Nox-type oxidases, intracellular peroxide, and ERK MAP kinase, respectively, on the enhancement of contraction to 30 mM KCl caused by stretch or lactate. The change in force developed by the Post (in Figs. 3A and 4A) contraction to 30 mM KCl was compared with the initial Pre contraction and expressed as a percentage. In other studies, arterial rings precontracted with 30 mM KCl were exposed to peroxide (100 µM or 1 mM H2O2) and the response compared in the presence or absence of the mitogen-activated protein kinase kinase (MEK) inhibitor 10 µM PD-98059.



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Fig. 3. Influence of Nox-derived reactive oxygen species (ROS), cytosolic NAD(H) redox and ERK activation in a stretch-mediated enhancement of pulmonary arterial force generation. A: representative tracing from the Powerlab Chart system demonstrating force development to KCl in bovine pulmonary arterial rings. Force development is measured (in g) increasing along the y-axis as a function of time (in min). Note the indicated additions of drugs at point X along the time frame of the protocol, as well as the maintained exposure to 20 g of passive stretch for 20 min between "Pre," the prestretch control contraction to 30 mM KCl, and "Post," the poststretch contraction to 30 mM KCl. The difference is then calculated and expressed as a percentage of the prestretch contraction to assess changes in contractile function. B and C: summary data (n = 8) of force generation to 30 mM KCl and ERK phosphorylation, respectively, from bovine pulmonary arterial ring segments in the absence or presence of combinations of stretch treatment protocol and/or pretreatment with drugs (10 µM PD-98059, 0.1 mM apocynin, or 100 µM ebselen) or redox metabolites (10 mM lactate or 10 mM pyruvate).

 


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Fig. 4. Influence of Nox-derived ROS, hypoxia, and ERK activation on a lactate-mediated enhancement of pulmonary arterial force generation. A: representative tracing from the Powerlab Chart system demonstrating force development to KCl in bovine pulmonary arterial rings. Force development is measured in grams increasing along the y-axis as a function of time (in min). Note the indicated additions of drugs at point X along the time frame of the protocol, followed by the 30-min period of incubations in the absence or presence of 10 mM lactate between Pre, the preincubation control contraction to 30 mM KCl, and Post, the postincubation contraction to 30 mM KCl in the absence or presence of lactate, drugs, and/or hypoxia. The difference is then calculated and expressed as a percentage of the preincubation contraction to assess alterations in contractile function. B and C: summary data for changes in force generation and ERK phosphorylation, respectively (n = 8). Bovine pulmonary arterial ring segments were incubated for 30 min in the absence or presence of 10 mM lactate and/or a 15-min preincubation with hypoxia or drugs that inhibit either the ERK pathway, 10 µM PD-98059, or Nox oxidases, 100 µM apocynin (n = 8).

 
DPI (1 µM), hypoxia (95% N2-5% CO2), 10 µM PD-98059, and 1 µM PP2 were used to investigate the roles of flavoprotein oxidases, oxygen-dependent processes, MEK, and src kinase, respectively, in the mechanism of a slowly developing contraction caused by low concentrations of diamide. In brief, vessels were prepared and exposed as described above, allowed to equilibrate (and preincubated for 15 min with drugs where applicable), and exposed to 1 µM diamide for 60 min. Change in force was recorded over the time period, and vessels were rapidly frozen in liquid nitrogen at the end of the time course to be analyzed further via Western blot analysis.

Detection of changes in superoxide. Changes in superoxide levels in endothelium-denuded pulmonary arteries resulting from exposure to the stretch protocol were quantified by the methods previously described (18, 21) for measurement of force in a single photon-counting chemiluminescence detection apparatus constructed in a light-tight box. In these experiments, pulmonary artery rings were incubated in Krebs-bicarbonate buffer containing 5 µM lucigenin in a cuvette continuously gassed with 21% O2. The chemiluminescence observed at 20 and 60 min after exposure to stretch was reported as a percentage of the chemiluminescence observed before stretch in a protocol that included measurements of contractions to 30 mM KCl before and 60 min after exposure to the stretch protocol. Pulmonary arteries exposed to the same protocol omitting exposure to stretch were employed as controls for examining the effects of stretch.

Western blotting. Studies comparing force generation to ERK phosphorylation utilized arteries that were rapidly removed, blotted, and frozen for Western blot analysis usually during the second 30 mM KCl contractile response after force had reached a steady state under the conditions reported in RESULTS. Frozen arterial segments were pulverized under liquid nitrogen and placed in a homogenization buffer [60 mM Tris, 10 mM EGTA, 2 mM EDTA, protease and phosphatase inhibitors (Sigma), pH 7.5]. The tubes were centrifuged, the supernatant was isolated, and protein levels were assayed by a modified Bradford method (2) for each sample. Ten micrograms of each sample were loaded and run on 12% SDS-PAGE gels, transferred to supported nitrocellulose membranes, subsequently exposed to primary and secondary antibodies in 5% milk/TBS-Tween buffers, and detected by Amersham Biosciences ECL detection kits on autoradiography film. Densitometric analysis via the Kodak 1D system was used to quantitate protein levels. Phosphorylated ERK measurements reported in RESULTS were normalized to the total ERK form measured after stripping and reblotting the gels and then expressed as percentages of the control condition used in each experimental group. The detection of total ERK did not appear to be altered by any of the conditions examined. Total and phosphorylated forms of ERK antibodies were from Sigma/RBI (St. Louis, MO).

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


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
H2O2 elicits pulmonary artery contraction through activating ERK MAP kinase. Endothelium-denuded bovine pulmonary arteries precontracted with KCl were reported in a previous study to show minimal relaxation to H2O2 and an increase in force when they were exposed to 1 mM peroxide (3). Figure 1A contains a typical Western analysis showing that ERK MAP kinase phosphorylation appears increased in endothelium-denuded bovine pulmonary arteries precontracted with 30 mM KCl when they are exposed to 0.1 and 1 mM H2O2. As shown in Fig. 1B, attenuating the activation of ERK with 10 µM PD-98059, an inhibitor of its phosphorylation by MEK, promotes relaxation to 0.1 mM H2O2 and converts contraction to the 1 mM dose of H2O2 to a relaxation. Summary data in Fig. 1C indicate that 1 mM H2O2 increased the phosphorylation of ERK compared with vessels not exposed to H2O2, whereas vessels pretreated with PD-98059 and then exposed to 1 mM peroxide demonstrated levels of ERK phosphorylation that were significantly below that of control vessels. Although the 0.1 mM dose of H2O2 appears to cause a modest relaxation and an increase in ERK, these responses were somewhat variable and did not reach statistical significance.



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Fig. 1. Role of ERK in the response of pulmonary arteries to peroxide (H2O2). A: Western blot showing 0.1 and 1 mM H2O2 increase ERK phosphorylation (pERK) over baseline levels seen in the presence of 30 mM KCl, together with measurements of total ERK from the same gels. B: summary data for the effects of 100 µM and 1 mM H2O2 on the change in force generation to 30 mM KCl. The presence of the ERK pathway inhibitor 10 µM PD-98059 promoted relaxation to 100 µM H2O2, whereas the contraction to the higher dose of H2O2 was converted to a relaxation by ERK pathway inhibition (n = 6). C: changes in ERK phosphorylation caused by exposure of bovine pulmonary arteries to 100 µM and to 1 mM H2O2 in the presence and absence of 10 µM PD-98059 (PD) are compared with untreated, time-control vessels (Control) after normalizing to the measured amount of total ERK (n = 6).

 
Alteration of cytosolic NAD(H) redox through the LDH reaction modulates ERK activity. Since we previously provided evidence that bovine pulmonary arteries possess an oxidase generating superoxide and relaxant levels of H2O2, which is controlled by NADH availability, based on studying the influence of modulating cytosolic NAD(H) redox with lactate and pyruvate through the LDH reaction (9, 22), we hypothesized that modulating this redox system could function to regulate the control of ERK by endogenous H2O2. As shown in Fig. 2, exposure of bovine pulmonary arteries to 10 mM lactate or 10 mM pyruvate was observed to cause changes in the levels of ERK phosphorylation in vessels that were freeze-clamped after a 30-min incubation period and subsequently analyzed by Western blot analyses. The data in Fig. 2 show a concentration-dependent increase in the levels of phosphorylated ERK by lactate and a decrease or inhibition of phosphorylation by pyruvate.



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Fig. 2. Influence of lactate and pyruvate on ERK phosphorylation. Lactate and pyruvate have apparently dose-dependent, opposing effects on ERK phosphorylation as seen in A, which contains a representative Western blot of increasing doses of lactate and pyruvate, and in summary data shown in B (n = 8). Lac, lactate; Pyr, pyruvate; Ctrl, control.

 
Stretch causes enhanced force generation and ERK activation in pulmonary arteries. Our initial studies on the effects of stretch were performed in the coronary artery (21), and we were interested to determine whether a similar phenomenon is present in the pulmonary circulation as well. A 20-min period of stretch was chosen for the study using the protocol shown in Fig. 3A, because this stretch treatment caused prolonged increases in superoxide for over a 1-h period as determined by lucigenin chemiluminescence, similar to the response previously described in bovine coronary arteries (21). The level of superoxide detected by measuring chemiluminescence derived from 5 µM lucigenin was significantly increased to 148 ± 9% (n = 4) of the prestretch chemiluminescence at 20 min after the release of stretch and remained elevated (171 ± 42%) 60 min after the release of stretch, whereas superoxide levels were not significantly elevated in control arteries (108 ± 16%, n = 4) that were not exposed to the stretch treatment at the time point corresponding to 60 min. Time-controlled contractions in unstretched pulmonary arterial segments developed 104 ± 6% (Fig. 3B) and 107 ± 4% (n = 4) of the prestretch 30 mM KCl contraction at the 20- and 60-min time points when re-exposed to 30 mM KCl at these time points. In arteries exposed to stretch, contractile force to 30 mM KCl averaged ~70% greater than that seen in prestretched controls at the 20-min time point (Fig. 3B) and was 71 ± 11% greater (P < 0.05, n = 4) than the control contraction at 60 min after exposure to stretch. Vessels stretched while being continuously gassed under nitrogen did not demonstrate an enhancement in contractile function to 30 mM KCl (95 ± 11% of control), yet interestingly upon reoxygenation and repeat of the stretch under normal aeration, the stretch response returned (166 ± 17% of control), suggesting a role for oxygen in the changes that are observed. Furthermore, Western analysis of stretched pulmonary arteries demonstrated a twofold increase in the levels of phosphorylated ERK (see Fig. 3C).

Stretch-enhanced contractile function is dependent on NADH oxidase and ERK MAP kinase signaling. To develop evidence for a role of NADH oxidase and the ERK pathway in the response to stretch, we incubated vessels with the Nox oxidase inhibitor 100 µM apocynin, an enhancer of intracellular peroxide metabolism, 100 µM ebselen, 10 mM lactate, or 10 mM pyruvate. Additionally, to confirm the dependence of Nox oxidase-derived reactive oxygen species (ROS) signaling specifically through the ERK MAP kinase pathway, we exposed another subset of vessels to the MEK inhibitor 10 µM PD-98059. All vessels were then subjected to the passive stretch protocol. Figure 3, B and C, contains summary data for the enhancement of force development and the increase in ERK MAP kinase phosphorylation caused by stretch. The increase in force generation was attenuated 88% by PD-98059 and was not observed in the presence of apocynin, ebselen, and pyruvate. With the exception of ebselen, these agents did not have a statistically significant effect on force generation to 30 mM KCl in unstretched, incubated time controls. Ebselen may be increasing force in these arteries by removing a basal peroxide-mediated relaxation, which we have characterized in previous studies (19, 22). Vessels incubated with lactate retained the stretch-induced enhancement in contractile function and had a statistically significant increase in the contractile response to 30 mM KCl in incubated time-control arteries not exposed to stretch.

The origins of increases in ERK phosphorylation associated with enhanced force generation elicited by exposure to the stretch protocol were studied in Fig. 3C. In a pattern similar to that of the force changes (compare with Fig. 3B), the approximate doubling of ERK phosphorylation caused by stretch is prevented by PD-98059, apocynin, ebselen, and pyruvate. Additionally, PD-98059 and pyruvate decreased ERK phosphorylation, whereas lactate increased phosphorylation by ~75% above baseline levels seen in control incubated tissues not exposed to the stretch protocol. Although ebselen appeared to be increasing ERK phosphorylation, the changes observed were not statistically significant.

Role of lactate and pyruvate modulation of ERK in pulmonary arterial contractile function. As demonstrated in Figs. 2 and 3, it appears that lactate increases ERK phosphorylation and force development by KCl in a manner consistent with its modulating the availability of NADH. To investigate whether NADH oxidase activity is important in responses elicited by the experimental addition of lactate, we utilized preincubation with 0.1 mM apocynin to investigate the role of a Nox-type oxidase. Although apocynin had no significant effect on force generation or ERK phosphorylation in control vessels, as seen in Fig. 4, B and C, it abolished the lactate-induced increases in both ERK phosphorylation and contractile function.

We have previously demonstrated that ROS activation of the ERK family of MAP kinases is involved in the changes in contractility to stretch. To determine whether this pathway is involved in the redox-modulated changes as well, the responses to lactate were examined in the presence of hypoxia, 100 µM ebselen, and under conditions where the activation of ERK by MEK was inhibited with 10 µM PD-98059. Hypoxia increased force without affecting ERK phosphorylation, and lactate did not alter contraction to 30 mM KCl or ERK phosphorylation under hypoxia (Fig. 4, B and C). The contraction to 30 mM KCl (122 ± 13% of control, n = 12) and level of ERK phosphorylation (ERK-Pi = 132 ± 29% of control, n = 9) observed in the presence of 100 µM ebselen were not significantly altered by 10 mM lactate in the presence of ebselen [KCl = 124 ± 15% (n = 12), ERK-Pi = 176 ± 32% (n = 9)]. The data in Fig. 4 also demonstrate that PD-98059 attenuates the lactate-induced increase in force generation and ERK phosphorylation. Neither the MEK inhibitor nor the vehicle 0.1% DMSO significantly altered the contractile response to 30 mM KCl in arteries that were not exposed to redox modulation (data not shown).

Thiol oxidation activates an ERK-mediated contraction. The role of thiol modification as the potential protein-oxidant interaction or "entry point" into the kinase signaling cascade was further investigated. Vessels were incubated with the thiol oxidant 1 µM diamide for 5, 30, and/or 60 min, freeze clamped, and analyzed for changes in ERK phosphorylation. There is a rapid activation of the ERK MAP kinase pathway at 5 min (175 ± 21%) that appears to remain maximally elevated at the 60-min time point (206 ± 24% of time control, n = 6). Because preliminary experiments suggested the thiol modification leading to ERK activation appeared to be oxidase independent, because it was not prevented by inhibition of pulmonary artery flavoprotein containing oxidases with 1 µM diphenyliodonium (19) or by a hypoxic atmosphere of 95% N2-5% CO2 (data not shown), we attempted to localize the thiol modification site pharmacologically. As seen in Fig. 5, A and C, the increase in ERK phosphorylation by diamide at the 60-min time point was attenuated by the MEK inhibitor PD-98059, but not by the src inhibitor PP2 (1 µM), suggesting the existence of a thiol redox-sensitive site between these two kinases, which are thought (21) to be on the pathway regulating the activation of ERK by stretch and peroxide. Coinciding with this increase in ERK phosphorylation is a small, but significant diamide-elicited contraction (3.6 ± 0.3 g, n = 11 compared with force changes 0.8 ± 0.4 g, n = 11 in baseline time controls) that develops over the 60-min period after the addition of diamide. This contraction is attenuated by the ERK pathway inhibitor PD-98059 but not by inhibition of src signaling with PP2. Summary data for these changes in force are shown in Fig. 5B.



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Fig. 5. Activation of an ERK-mediated contraction by the thiol oxidant diamide. A: representative Western analysis showing the increase in ERK phosphorylation by exposure to 1 µM diamide for 60 min, which is prevented by the MEK inhibitor PD-98509, but not by the src inhibitor 1 µM PP2. B and C: summary data for the contraction in measured grams of force that develops after a 60-min incubation with diamide and the changes in ERK phosphorylation, respectively. Pulmonary arterial ring segments were preincubated for 15 min in the absence or presence of 10 µM PD-98059 or 1 µM PP2 and then exposed to 1 µM diamide for 60 min. In B, the changes in force caused by a 60-min exposure to 1 µM diamide are compared with a time control of pulmonary arterial ring segments, which were not treated with diamide (n = 11). At the end of the 60-min time period, vessels were frozen and analyzed by Western analysis for changes in ERK phosphorylation (n = 6). Dia, diamide; PP2, src kinase inhibitor.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of this study provide evidence for the presence in bovine pulmonary arteries of an ERK MAP kinase-mediated contractile mechanism that is regulated by exogenous and endogenously formed H2O2. This mechanism appears to function in a manner that opposes a previously studied relaxing mechanism to peroxide that appears to function through cGMP. The effects of lactate and pyruvate on modulating the basal levels of ERK MAP kinase phosphorylation and force generation are consistent with a role for H2O2 derived from an oxidase whose activity is controlled by cytosolic NADH redox in regulating the ERK MAP kinase signaling system through the hypothesized mechanism shown in Fig. 6. In this mechanism, the metabolism of lactate by the LDH reaction increases the availability of cytosolic NADH as a substrate for Nox-mediated peroxide generation, and this activates an ERK-mediated enhancement of force generation. In addition, bovine pulmonary arteries undergo a stretch-induced enhancement of contraction that appears to involve a Nox oxidase-dependent stimulation of ERK MAP kinase based on the observation of an inhibition to these responses by apocynin and PD-98059. Pyruvate was able to reverse this passive stretch-mediated enhancement of force. Because the thiol oxidant diamide activated a contraction through the ERK MAP kinase system that seemed to be independent of tissue oxidase activity, peroxide may stimulate this contractile mechanism through a process involving thiol oxidation.



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Fig. 6. Hypothesized pathway through which cytosolic NAD(H) redox and stretch regulate pulmonary arterial force through regulation of ERK MAP kinase. On the basis of previous work (21), it is hypothesized that stretch causes an increased apocynin-inhibited translocation of cytosolic subunits that activate superoxide production by a Nox oxidase. Data in the present study with lactate and pyruvate suggest that the activity of this oxidase is influenced by cytosolic NAD(H) through modulating the lactate dehydrogenase (LDH) reaction. The production of superoxide (O2·) and its subsequent dismutation to H2O2 leads to an increased activation of the ERK MAP kinase through a src-epidermal growth factor-mitogen-activated protein kinase kinase (EGFR/MEK) signaling pathway (21). Thiol oxidation with diamide activates ERK at a location downstream of src and upstream of the MEK, which remains to be elucidated. Signaling through the well defined ERK MAP kinase pathway can then lead to phosphorylations on regulators of the contractile apparatus, such as caldesmon and calponin. The phosphorylation of these proteins is thought to promote their dissociation from actin and an enhancement of force through altered cross bridge cycling frequency and contractile efficiency (17, 20). P, phosphorylation.

 
Peroxide regulates pulmonary artery force through simultaneously activating both relaxing and ERK-mediated contracting mechanisms. In this study we developed evidence for a role for stimulation of ERK-mediated contraction as an explanation for the previously observed (3) biphasic concentration-dependent relaxation-contraction response of precontracted endothelium-denuded bovine pulmonary arteries elicited by exposure to H2O2. KCl was selected in the present study as the contractile agent because it minimizes expression of relaxation to H2O2. Under increasing concentrations of peroxide there appears to be an activation of an alternative contractile pathway mediated through ERK MAP kinase, as demonstrated in Fig. 1 by the observation of a relaxation at the lower dose and reversal from contraction to relaxation in the higher dose of peroxide when the activation of ERK is inhibited. The role of ERK in this response of pulmonary arteries is like the recently reported (21) ERK-mediated contractile component of a similar biphasic response of bovine coronary arteries caused by exposure to increasing doses of H2O2. Relaxing mechanisms including stimulation of soluble guanylate cyclase and cGMP (3), opening potassium channels (27), and additional redox mechanisms controlling calcium caused by oxidizing cytosolic NADPH (8) could be contributing factors to the relaxing component of the response of pulmonary arteries to peroxide. Stimulation of tyrosine kinases has been reported to contribute contractile responses elicited by peroxide (11). Because multiple tyrosine kinases participate in the src-EGF receptor pathway previously identified in vascular tissue linking peroxide to the stimulation of ERK, mechanisms examined in this study could be an important contributing factor to processes through which redox regulation of tyrosine phosphorylation contributes to the regulation of force in pulmonary arteries. Force regulation by MAP kinase systems has been a topic of much research and debate, and there is evidence linking ERK to control of the contractile apparatus in smooth muscle through regulation of caldesmon and calponin (17, 20).

Cytosolic NADH regulates force generation through a Nox oxidase-mediated activation of ERK. The data in this study provide evidence that lactate and pyruvate have opposing effects on ERK phosphorylation that can be associated with their effects on the regulation of force generation. The data in Figs. 3 and 4 provide evidence that prolonged incubation with lactate increases force through stimulating the ERK MAP kinase pathway. On the basis of our previous work demonstrating that lactate causes a rapidly developing relaxation of pulmonary arteries through a mechanism that appears to involve endogenous H2O2 generation, it is likely that lactate is also simultaneously activating a relaxing mechanism that prevents the full expression of its ERK-mediated contracting mechanism in a manner similar to the effects of exposing pulmonary arteries to exogenous H2O2. Because lowering basal levels of ERK activation with pyruvate did not depress force, it is likely that the basal level of phosphorylation of ERK is below the threshold for influencing the contraction to 30 mM KCl. Our studies in bovine coronary arteries (21) were also consistent with force generation to 30 mM KCl not being influenced by the basal levels of ERK phosphorylation, whereas increased ERK activation by peroxide or stretch promoted an enhancement of force in a manner similar to peroxide and lactate observed in the present study. However, when stretch promoted an increase in activation of ERK, the inhibition of this activation of ERK by pyruvate was associated with an attenuation of the stretch-induced increase in force. Because the increase in force and ERK activation caused by lactate were prevented by an inhibitor of Nox-type oxidases, the increase in cytosolic NADH that is hypothesized to be caused by lactate is likely to be a controlling factor in the generation of peroxide, which is thought to initiate the activation of ERK.

Stretch activates an ERK-mediated contraction-enhancing mechanism in pulmonary arteries that appears to be regulated by the availability of cytosolic NADH. Stretch was observed to cause an enhancement of contraction in bovine pulmonary arteries through a mechanism that appears to involve activation of a Nox oxidase-mediated stimulation of ERK MAP kinase in a manner similar to the response to stretch that was observed in bovine coronary arteries (21). This is based on the studies in bovine coronary arteries showing that the response to stretch was associated with an apocynin-inhibited membrane binding of the p47phox Nox oxidase subunit (21) and by observing in pulmonary arteries that the stretch-induced enhancement of force and ERK phosphorylation were attenuated by apocynin, presumably through a similar inhibition of Nox-activation by its p47phox subunit. Stretch increased the detection of superoxide by lucigenin chemiluminescence, and increasing the intracellular metabolism of peroxide with ebselen also appeared to prevent observation of the ERK-associated force enhancing effects of stretch. The role of ERK in the response is supported by stretch causing an increase in its phosphorylation and by inhibition of the stretch induced increases in force generation and ERK phosphorylation by the MEK inhibitor PD-98059. On the basis of the known actions of pyruvate on lowering cytosolic NADH levels through its metabolism by the LDH enzyme, the availability of cytosolic NADH is potentially a key factor in controlling the peroxide-generating activity of the Nox-oxidase stimulated by stretch. This is based on the observations that pyruvate selectively inhibited increases in ERK phosphorylation and the ERK-associated increase in force caused by stretch, without its directly altering contraction to 30 mM KCl. Pyruvate has metabolic antioxidant actions potentially originating from preventing the oxidation of NADPH and glutathione (13), which may also contribute its observed effects on decreasing ERK phosphorylation. The actions of lactate on the stretch response are more difficult to interpret, because lactate appears to directly activate components of the ERK-linked signaling mechanism stimulated by stretch presumably through its direct effects on increasing endogenous peroxide generation by its functioning to provide NADH as a substrate for the Nox oxidase involved. When analyzing the interactions of lactate with the response to stretch one needs to also consider that lactate may also be simultaneously activating a peroxide-mediated relaxing mechanism (4, 7, 22) by providing NADH to Nox enzymes, which may not be regulated by stretch, and stretch may also activate other signaling mechanisms such as protein kinase C (25), which may function together with ERK activation to enhance force generation. Overall, the data are consistent with stretch activating an ERK-mediated contraction-enhancing mechanism in pulmonary arteries through stimulating a Nox oxidase that appears to be controlled by the redox status of cytosolic NAD(H).

Thiol redox changes may stimulate peroxide-independent mechanisms of ERK activation. The process through which peroxide interacts with the pathway linked to ERK activation is not known, and it could be hypothesized to originate from a peroxide-induced change in thiol redox. Because we observed that, in the bovine pulmonary arteries examined in this study, very low doses of diamide activated a slowly developing contraction, which was attenuated by MEK inhibition, we began to investigate if this drug provides evidence on how peroxide is activating ERK. Our previous studies (21) suggested that the src-EGF receptor region of the pathway activating ERK shown in Fig. 6 is the earliest step detected on the pathway to ERK activation by stretch after the formation of peroxide by oxidase activation. Thus after confirming that diamide was not functioning through a peroxide-dependent pathway by determining that its contractile response was not attenuated by preventing peroxide generation with hypoxia and inhibition of essentially all flavoprotein oxidases with DPI, we examined whether inhibition of src with PP2 altered the contraction to diamide. Since it did not, a process between the activation of src and the control of the phosphorylation of ERK by MEK is being activated by the thiol oxidant diamide. Because diamide has many potential actions that could function to influence the ERK pathway, including inhibiting thiol-containing protein phosphatases (6, 14, 16, 23), additional studies are needed to elucidate how diamide is activating ERK. Although the actions of diamide observed in this study do not appear to provide new information on how peroxide is activating ERK, they raise the possibility that thiol redox-regulated processes may have additional sites through which they regulate ERK activation.

Role of peroxide regulation of ERK in the control of pulmonary vascular function. The ERK MAP kinase system is a regulator of both pulmonary endothelial cell function (1, 13, 15, 26) and vascular smooth muscle growth (5, 10, 12, 24, 28), which could be of importance in remodeling that occurs in pulmonary hypertension. Observations made in this study demonstrate that peroxide causes pulmonary arterial smooth muscle contraction through activation of ERK and that cytosolic NAD(H) redox is a key regulator of the basal activity of this system. The contraction to hypoxia in bovine pulmonary arteries is not associated with increases in ERK phosphorylation, and hypoxia was observed to inhibit the lactate-elicited activation of this system. Exposure of pulmonary arteries to a brief period of stretch, which might occur under pulmonary hypertensive conditions, stimulated the ERK pathway through the effects of stretch on oxidase activation, resulting in a subsequent enhancement of force generation. Although the role of ERK stimulation by the redox and stretch-activated processes examined in this study are not understood, it is likely that these are fundamental regulatory systems that contribute to pulmonary vascular control processes associated with pathophysiological situations that alter oxidant production and redox, as well as conditions such as increased pressure, which would activate the ERK-mediated response to stretch.


    ACKNOWLEDGMENTS
 
We thank Dr. Pawel M. Kaminski for conducting the experiments detecting superoxide and Heather Berman for running some of the Western analyses measuring changes in ERK. This work was supported by United States Public Health Service Grants HL-31069, HL-43023, HL-63182, and HL-66331.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. S. Wolin, Dept. of Physiology, Basic Science Bldg., Rm. 604, New York Medical College, Valhalla, NY 10595 (E-mail: mike_wolin{at}nymc.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.


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