Evidence for the role of p38 MAP kinase in hypoxia-induced pulmonary vasoconstriction

Mitogen-activated protein (MAP) kinases regulate smooth muscle cell contraction. Hypoxia contracts pulmonary arteries by mechanisms that are incompletely understood. We hypothesized that hypoxic contraction of pulmonary arteries involves activation of the MAP kinases. To test this hypothesis, we studied the effects of SB-202190, a p38 MAP kinase inhibitor, PD-98059 and UO-126, two structurally different MEKK inhibitors, and anisomycin, a stimulator of p38 MAP kinase on acute hypoxia-induced contraction in rat conduit pulmonary artery rings precontracted with phenylephrine or KCl. Hypoxia induced a transient contraction, followed by a relaxation, and then a slowly developing sustained contraction. Hypoxia also significantly increased phosphorylation of p38 MAP kinase. SB-202190 did not affect the transient phase but abrogated the sustained phase of hypoxic contraction, whereas anisomycin enhanced both phases of contraction. SB-202190 also attenuated and anisomycin enhanced the phenylephrine-induced contraction. In contrast, PD-98059 and UO-126 had minimal effects on either hypoxic or phenylephrine-induced contraction. None of the treatments modified KCl-induced contraction. We conclude that p38, but not the ERK1/ERK2 MAP kinase pathway, mediates the sustained phase of hypoxic contraction in isolated rat pulmonary arteries.     

Nonspecific inhibition of myogenic tone by PD98059, a MEK1 inhibitor, in rat middle cerebral arteries

Activation of MAP kinase kinase, also called ERK kinase (MEK), may lead to desinhibition of thin filament regulatory proteins and we therefore investigated the acute effects of the potent MEK inhibitor, PD98059 on the contractile properties of pressurized rat middle cerebral arteries. Cerebral arteries (diameter 100-150 microm) were mounted on a pressure myograph and PD98059 (10 microM, 40 microM) significantly inhibited (15% and 64%) myogenic tone (P < 0.001). At these concentrations, PD98059 also significantly reduced the vasopressin (0.1 microM)- and KCl (60 mM)-induced tone. Cumulative addition of exogenous Ca2+ (0.4-1.6 mM) increased myogenic tone to approximately 50% of constriction at 80 mmHg. This effect was inhibited by PD98059 (P < 0.001). These results demonstrate that pressure-induced myogenic tone is inhibited by PD98059 at the concentrations that have been reported to be selective for inhibition of MEK and the MAP kinase cascade. However, our results also demonstrate that PD98059 may have nonspecific effects on voltage-sensitive Ca2+ entry in vascular smooth muscle.     

Cytosolic NADPH may regulate differences in basal Nox oxidase-derived superoxide generation in bovine coronary and pulmonary arteries

Because systems controlled by basal NAD(P)H oxidase activity appear to contribute to differences in responses of endothelium-removed bovine coronary (BCA) and pulmonary (BPA) arteries to hypoxia, we characterized the Nox oxidases activities present in these vascular segments and how cytosolic NAD(P)H redox systems could be controlling oxidase activity. BPA generated approximately 60-80% more lucigenin (5 microM) chemiluminescence detectable superoxide than BCA. Apocynin (10 microM), a NAD(P)H oxidase inhibitor, and 6-aminonicotinamide (1 mM), a pentose phosphate inhibitor (PPP), both attenuated (approximately by 50-70%) superoxide detected in BPA and BCA. There was no significant difference in the expression of Nox2 or Nox4 mRNA or protein detected by Western blot analysis. NADPH and NADH increased superoxide in homogenates and isolated microsomal membrane fractions in a manner consistent with BPA and BCA having similar levels of oxidase activity. BPA had 4.2-fold higher levels of NADPH than BCA. The activity and protein levels of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting PPP enzyme generating cytosolic NADPH, were 1.5-fold higher in BPA than BCA. Thus BPA differ from BCA in that they have higher levels of G6PD activity, NADPH, and superoxide. Because both arteries have similar levels of Nox expression and activity, elevated levels of cytosolic NADPH may contribute to increased superoxide in BPA.     

Evidence for modulation of smooth muscle force by the p38 MAP kinase/HSP27 pathway

Mitogen-activated protein (MAP) kinases signal to proteins that could modify smooth muscle contraction. Caldesmon is a substrate for extracellular signal-related kinases (ERK) and p38 MAP kinases in vitro and has been suggested to modulate actin-myosin interaction and contraction. Heat shock protein 27 (HSP27) is downstream of p38 MAP kinases presumably participating in the sustained phase of muscle contraction. We tested the role of caldesmon and HSP27 phosphorylation in the contractile response of vascular smooth muscle by using inhibitors of both MAP kinase pathways. In intact smooth muscle, PD-098059 abolished endothelin-1 (ET-1)-stimulated phosphorylation of ERK MAP kinases and caldesmon, but p38 MAP kinase activation and contractile response remained unaffected. SB-203580 reduced muscle contraction and inhibited p38 MAP kinase and HSP27 phosphorylation but had no effect on ERK MAP kinase and caldesmon phosphorylation. In permeabilized muscle fibers, SB-203580 and a polyclonal anti-HSP27 antibody attenuated ET-1-dependent contraction, whereas PD-098059 had no effect. These results suggest that ERK MAP kinases phosphorylate caldesmon in vivo but that activation of this pathway is unnecessary for force development. The generation of maximal force may be modulated by the p38 MAP kinase/HSP27 pathway.     

Molecular regulation of NADPH oxidase 5 via the MAPK pathway

The mechanisms controlling the activity of NADPH oxidase 5 (Nox5) are unique in that they are independent of the protein: protein interactions that coordinate the activation of other Nox isoforms. Instead, the primary driving force for Nox5 activity is calcium. However, in a previous study we reported that the protein kinase C (PKC)-agonist PMA could induce a sustained activation of Nox5 that was independent of calcium changes. This apparent calcium-independent activation was found to be mediated by the PKC-dependent phosphorylation of specific serine and threonine residues on Nox5 which increased the calcium sensitivity of the enzyme and enabled activation at resting levels of calcium. However, the specific kinase(s) mediating the phosphorylation and activation of Nox5 are not known. As PKC can activate the MEK/ERK1/2 signaling pathway, we hypothesized that Nox5 is activated by the coordinated phosphorylation of both MAPK and PKC pathways. The inhibition of MEK1 using PD-98059 and U-0126 significantly reduced the phosphorylation and activity of Nox5 in response to PMA but not to the calcium-mobilizing stimulus ionomycin. Dominant negative MEK1 and knockdown of endogenous MEK1/2 using a specific small interfering RNA also inhibited Nox5 activity in response to PMA. The mutation of S498 to a nonphosphorylatable residue and to a lesser degree T494 blocked the ability of ERK to stimulate Nox5 activity. However, a constitutively active form of MEK1 failed to increase Nox5 activity in the absence of PMA stimulation. These results suggest that the MEK/ERK1/2 pathway is necessary but not sufficient to regulate the PMA-dependent activation of Nox5.     

MAPK and PKC activity are not required for H(2)O(2)-induced arterial muscle contraction

H(2)O(2)-induced pulmonary arterial smooth muscle (PASM) contractions are independent of Ca(2+) and myosin light chain phosphorylation. The purpose of this study was to determine whether mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1 and ERK2, or protein kinase C (PKC) activation is required for H(2)O(2)-induced contraction. Porcine PASM strips were stimulated with 1 mM H(2)O(2), 120 mM KCl, or 10 microM phorbol myristic acetate and freeze clamped at various times during the contractions. Changes in relative amounts of tyrosine/threonine phosphorylated MAPK compared with total MAPK were measured. MAPK tyrosine phosphorylation levels increased in correlation with tension development. However, 50 microM PD-98059, a MAPK/ERK kinase-MAPK kinase blocker, reduced MAPK phosphorylation below resting levels, even though the magnitude of the isometric tension development was unaltered. Freeze-clamped PASM strips were placed in a PKC activity assay buffer containing (32)P and CaCl(2) to measure the total myelin basic protein phosphorylation. The data show that: 1) the time courses of PKC activity and force produced in response to H(2)O(2) do not correlate, and 2) MAPK activation may be a concurrent event with, or a consequence of, tension development in response to a variety of agonists but is not responsible for contractions to H(2)O(2), high K(+), or phorbol esters.     

HIV-1 Tat activates dual Nox pathways leading to independent activation of ERK and JNK MAP kinases

Human immunodeficiency virus, type 1 Tat is known to exert pleiotropic effects on the vascular endothelium through mitogen-activated protein (MAP) kinases, although the signaling pathways leading to MAP kinase activation are incompletely understood. We focused on proximal pathways potentially governing downstream MAP kinase activity by Tat. Within 2 min, Tat activated both Ras and Rho GTPases in endothelial cells, leading to ERK phosphorylation by 10 min. Notably, Rac1 was necessary for downstream activation of RhoA and both Rac1 and RhoA acted upstream of the Ras/ERK cassette. Antioxidants and the oxidase inhibitor diphenylene iodonium blocked ERK phosphorylation, but specific interference with the canonical Nox2 oxidase had no effect on ERK. Instead, knock down of the novel oxidase Nox4 completely suppressed Tat-dependent Ras and ERK activation downstream of Rac1 and RhoA. Conversely, interference with Rac1, PAK1, and Nox2 blocked JNK phosphorylation, whereas RhoA(N19) and Nox4 knock down did not. Further, knock down of Nox2, but not Nox4, blocked Tat-induced cytoskeletal rearrangement, whereas knock down of Nox4, but not Nox2, blocked Tat-dependent proliferation. Rac1, therefore, bifurcates Tat signaling, leading to concurrent but separate Nox4-dependent Ras/ERK activation, and Nox2-dependent JNK activation. Tat signaling, therefore, provides an example of Nox-specific differential control of MAP kinase pathways.     

Mechanical stretch enhances the expression of resistin gene in cultured cardiomyocytes via tumor necrosis factor-alpha

The heart is a resistin target tissue and can function as an autocrine organ. We sought to investigate whether cyclic mechanical stretch could induce resistin expression in cardiomyocytes and to test whether there is a link between the stretch-induced TNF-alpha and resistin. Neonatal Wistar rat cardiomyocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation at 60 cycles/min. Cyclic stretch significantly increased resistin protein and mRNA expression after 2-18 h of stretch. Addition of PD-98059, TNF-alpha antibody, TNF-alpha receptor antibody, and ERK MAP kinase small interfering RNA 30 min before stretch inhibited the induction of resistin protein. Cyclic stretch increased, whereas PD-98059 abolished, the phosphorylated ERK protein. Gel-shift assay showed a significant increase in DNA-protein binding activity of NF-kappaB after stretch, and PD-98059 abolished the DNA-protein binding activity induced by cyclic stretch. DNA binding complexes induced by cyclic stretch could be supershifted by p65 monoclonal antibody. Cyclic stretch increased resistin promoter activity, whereas PD-98059 and p65 antibody decreased resistin promoter activity. Cyclic stretch significantly increased TNF-alpha secretion from myocytes. Recombinant resistin protein and conditioned medium from stretched cardiomyocytes reduced glucose uptake in cardiomyocytes, and recombinant small interfering RNA of resistin or TNF-alpha antibody reversed glucose uptake. In conclusion, cyclic mechanical stretch enhances resistin expression in cultured rat neonatal cardiomyocytes. The stretch-induced resistin is mediated by TNF-alpha, at least in part, through ERK MAP kinase and NF-kappaB pathways. Glucose uptake in cardiomyocytes was reduced by resistin upregulation.     

The NADPH oxidase inhibitors iodonium diphenyl and cadmium sulphate inhibit hypoxic pulmonary vasoconstriction in isolated rat pulmonary arteries

Interest surrounds the role of an NADPH oxidase-like enzyme in hypoxic pulmonary vasoconstriction (HPV). We have studied the effects of the NADPH oxidase inhibitors iodonium diphenyl (ID) and cadmium sulphate (CdSO4) upon HPV of isolated rat pulmonary arteries (n = 73, internal diameter 545 +/- 23 microm). Vessels were preconstricted with prostaglandin F2alpha (PGF2alpha, 0.5 or 5 microM) prior to a hypoxic challenge. ID (10 or 50 microM), CdSO4 (100 microM) or vehicle (50 microl) was added for 30 min before re-exposure to PGF2alpha and hypoxia. ID and CdSO4 significantly inhibited HPV. In vessels preconstricted with 5 microM PGF2alpha, ID (10 and 50 microM) reduced HPV from 37.4 +/- 5.6 % to 9.67 +/- 4.4 % of the contractile response elicited by 80 mM KCl (P<0.05) and from 30.1 +/- 5.0 % to 0.63 +/- 0.6% 80 mM KCl response (P<0.01), respectively. CdSO4 (100 microM) reduced HPV from 29.4 +/-4.0 % to 17.1 +/- 2.2% 80 mM KCl response (P<0.05). In vessels preconstricted with 0.5 microM PGF2alpha, ID (10 and 50 microM) reduced HPV from 16.0 +/- 3.15% to 3.36 +/- 1.44 % 80 mM KCl response (P<0.01) and from 15.0 +/- 1.67 % to 2.82 +/- 1.40 % 80 mM KCl response (P<0.001), respectively. Constriction to PGF2alpha was potentiated by ID. ID and CdSO4, at concentrations previously shown to inhibit neutrophil NADPH oxidase, attenuate HPV in isolated rat pulmonary arteries. This suggests that an NADPH oxidase-like enzyme is involved in HPV and could act as the pulmonary oxygen sensor.     

Dual signaling by the alpha(v)beta(3)-integrin activates cytosolic PLA(2) in bovine pulmonary artery endothelial cells

Vitronectin, which ligates the alpha(v)beta(3)-integrin, increases both lung capillary permeability and lung endothelial Ca(2+). In stable monolayers of bovine pulmonary artery endothelial cells (BPAECs) viewed with confocal microscopy, multimeric vitronectin aggregated the apically located alpha(v)beta(3)-integrin. This caused arachidonate release that was inhibited by pretreating the monolayers with the anti-alpha(v)beta(3) monoclonal antibody (MAb) LM609. No inhibition occurred in the presence of the isotypic MAb PIF6, which recognizes the integrin alpha(v)beta(5). Vitronectin also caused membrane translocation and phosphorylation of cytosolic phospholipase A(2) (cPLA(2)) as well as tyrosine phosphorylation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) 2. The cPLA(2) inhibitor arachidonyl trifluoromethylketone, the tyrosine kinase inhibitor genistein, and the MAPK kinase inhibitor PD-98059 all blocked the induced arachidonate release. PD-98059 did not inhibit the increase of cytosolic Ca(2+) or cPLA(2) translocation, although it blocked tyrosine phosphorylation of ERK2. Moreover, although the intracellular Ca(2+) chelator MAPTAM also inhibited arachidonate release, it did not inhibit tyrosine phosphorylation of ERK2. These findings indicate that ligation of apical alpha(v)beta(3) in BPAECs caused ERK2 activation and an increase of intracellular Ca(2+), both conjointly required for cPLA(2) activation and arachidonate release. This is the first instance of a tyrosine phosphorylation-initiated "two-hit" signaling pathway that regulates an integrin-induced proinflammatory response.     

Roles for redox mechanisms controlling protein kinase G in pulmonary and coronary artery responses to hypoxia

We previously reported that isolated endothelium-removed bovine pulmonary arteries (BPAs) contract to hypoxia associated with removal of peroxide- and cGMP-mediated relaxation. In contrast, bovine coronary arteries (BCAs) relax to hypoxia associated with cytosolic NADPH oxidation coordinating multiple relaxing mechanisms. Since we recently found that H(2)O(2) relaxes BPAs through PKG activation by both soluble guanylate cyclase (sGC)/cGMP-dependent and cGMP-independent thiol oxidation/subunit dimerization mechanisms, we investigated if these mechanisms participate in BPA contraction and BCA relaxation to hypoxia. The contraction of BPA (precontracted with 20 mM KCl) to hypoxia was associated with decreased PKG dimerization and PKG-mediated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. In contrast, exposure of 20 mM KCl-precontracted endothelium-removed BCAs to hypoxia caused relaxation and increased dimerization and VASP phosphorylation. Depletion of sGC by organoid culture of BPAs with an oxidant of the sGC heme (10 μM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) increased aerobic force generation, decreased VASP phosphorylation, and inhibited further contraction to hypoxia and changes in VASP phosphorylation. Thiol reduction with dithiothreitol increased aerobic force in BPAs and decreased PKG dimerization, VASP phosphorylation, and the contraction to hypoxia. Furthermore, PKG-1α and sGC β(1)-subunit small interfering RNA-transfected BPAs demonstrated increased aerobic K(+) force and inhibition of further contraction to hypoxia, associated with an attenuation of H(2)O(2)-elicited relaxation and VASP phosphorylation. Thus, decreases in both a sGC/cGMP-dependent and a dimerization-dependent activation of PKG by H(2)O(2) appear to contribute to the contraction of BPAs elicited by hypoxia. In addition, stimulation of PKG activation by dimerization may be important in the relaxation of coronary arteries to hypoxia.     

Oxidant and redox signaling in vascular oxygen sensing mechanisms: basic concepts, current controversies, and potential importance of cytosolic NADPH

Vascular smooth muscle (VSM) derived from pulmonary arteries generally contract to hypoxia, whereas VSM from systemic arteries usually relax, indicating the presence of basic oxygen-sensing mechanisms in VSM that are adapted to the environment from which they are derived. This review considers how fundamental processes associated with the generation of reactive oxygen species (ROS) by oxidase enzymes, the metabolic control of cytosolic NADH, NADPH and glutathione redox systems, and mitochondrial function interact with signaling systems regulating vascular force in a manner that is potentially adapted to be involved in Po2 sensing. Evidence for opposing hypotheses of hypoxia, either decreasing or increasing mitochondrial ROS, is considered together with the Po2 dependence of ROS production by Nox oxidases as sensors potentially contributing to hypoxic pulmonary vasoconstriction. Processes through which ROS and NAD(P)H redox changes potentially control interactive signaling systems, including soluble guanylate cyclase, potassium channels, and intracellular calcium are discussed together with the data supporting their regulation by redox in responses to hypoxia. Evidence for hypothesized potential differences between systemic and pulmonary arteries originating from properties of mitochondrial ROS generation and the redox sensitivity of potassium channels is compared with a new hypothesis in which differences in the control of cytosolic NADPH redox by the pentose phosphate pathway results in increased NADPH and Nox oxidase-derived ROS in pulmonary arteries, whereas lower levels of glucose-6-phosphate dehydrogenase in coronary arteries may permit hypoxia to activate a vasodilator mechanism controlled by oxidation of cytosolic NADPH.     

Transforming growth factor-beta1 induces Nox4 NAD(P)H oxidase and reactive oxygen species-dependent proliferation in human pulmonary artery smooth muscle cells

Transforming growth factor-beta1 (TGF-beta1) is abundantly expressed in pulmonary hypertension, but its effect on the pulmonary circulation remains unsettled. We studied the consequences of TGF-beta1 stimulation on freshly isolated human pulmonary artery smooth muscle cells (HPASMC). TGF-beta1 initially promoted differentiation, with upregulated expression of smooth muscle contractile proteins. TGF-beta1 also induced expression of Nox4, the only NAD(P)H oxidase membrane homolog found in HPASMC, through a signaling pathway involving Smad 2/3 but not mitogen-activated protein (MAP) kinases. TGF-beta1 likewise increased production of reactive oxygen species (ROS), an effect significantly reduced by the NAD(P)H oxidase flavoprotein inhibitor diphenylene iodonium (DPI) and by Nox4 siRNAs. In the absence of TGF-beta1, Nox4 was present in freshly cultured cells but progressively lost with each passage in culture, paralleling a decrease in ROS production by HPASMC over time. At a later time point (72 h), TGF-beta1 promoted HPASMC proliferation in a manner partially inhibited by Nox4 small interfering RNA and dominant negative Smad 2/3, indicating that TGF-beta1 stimulates HPASMC growth in part by a redox-dependent mechanism mediated through induction of Nox4. HPASMC activation of the MAP kinases ERK1/2 was reduced by the NAD(P)H oxidase inhibitors DPI and 4-(2-aminoethyl)benzenesulfonyl fluoride, suggesting that TGF-beta1 may facilitate proliferation by upregulating Nox4 and ROS production, with transient oxidative inactivation of phosphatases and augmentation of growth signaling cascades. These findings suggest that Nox4 is the relevant Nox homolog in HPASMC. This is the first observation that TGF-beta1 regulates Nox4, with important implications for mechanisms of pulmonary vascular remodeling.     

Delayed arteriolar relaxation after prolonged agonist exposure: functional remodeling involving tyrosine phosphorylation

Although arteriolar contraction is dependent on Ca2+-induced myosin phosphorylation, other mechanisms including Ca2+ sensitization and time-dependent phenomena such as cytoskeletal and cellular reorganization may contribute to contractile events. We hypothesized that if arteriolar smooth muscle exhibits time-dependent behavior this may be manifested in differences in relaxation after short- and long-term exposure to contractile agonists. Studies were conducted in isolated arterioles pressurized to 70 mmHg. In initial experiments (n = 10), rate of relaxation was measured after acute (5 min) or prolonged (4 h) exposure to 5 microM norepinephrine (NE). Prolonged exposure to NE resulted in significantly (P < 0.05) increased time for relaxation in physiological salt solution. Rapid relaxation of vessels exposed to NE for 4 h was observed after superfusion with 0 mM Ca2+ buffer, indicating that the alteration in relaxation was reversible and Ca2+ dependent. A similarly impaired dilation was not observed with 4-h exposure to KCl (75 mM). To determine mechanisms contributing to the effects of prolonged NE exposure, studies were performed in the presence of the microtubule depolymerizing agent demecolcine (10 microM) or a series of tyrosine phosphorylation inhibitors. Although demecolcine caused significant vasoconstriction (P < 0.05) and potentiated NE vasoconstriction, it did not prevent the effect of long-term NE exposure on relaxation. Genistein, although having no effect on acute NE-induced contraction, concentration-dependently inhibited prolonged NE constriction. Similarly, Src (PP1) and p42/44 MAP kinase (PD-98059) inhibitors prevented maintenance of long-term NE contraction. The data indicate that prolonged exposure to NE induces biochemical alterations that impair relaxation after removal of the agonist. The contractile effects are Ca2+ dependent and involve tyrosine phosphorylation but do not appear to involve the polymerization state of the microtubule network.     

Caldesmon phosphorylation is catalyzed by two kinases in permeabilized and intact vascular smooth muscle

Smooth muscle contraction is initiated by myosin light chain (MLC) phosphorylation catalyzed by the Ca(2+) dependent MLC kinase. However, many aspects of smooth muscle contraction cannot be accounted for by MLC phosphorylation. One hypothesis that has received experimental support involves the thin filament protein caldesmon. Caldesmon inhibits myosin ATPase activity; phosphorylation of caldesmon relieves this inhibitory effect. The primary candidates for catalysis of caldesmon phosphorylation are the p42/p44 ERK MAP kinases. However, we and others have shown that inhibition of the ERK MAP kinases has no effect on many smooth muscles. The goal of this study was to determine if evidence for a second endogenous caldesmon kinase may be obtained. We used Triton X-100 skinned and intact tissues of the swine carotid artery to address this goal. Caldesmon phosphorylation was evident in resting and Ca(2+) stimulated Triton X-100 skinned fibers. Ca(2+)-dependent caldesmon phosphorylation was partially sensitive to the ERK MAP kinase inhibitor PD98059, whereas all caldesmon phosphorylation was sensitive to the general kinase inhibitor, staurosporine. Histamine increased caldesmon phosphorylation levels in intact swine carotid artery, which was sensitive to both PD98059 and staurosporine. Histamine increased ERK MAP kinase activity, which was reversed by PD98059, staurosporine, and EGTA. Histamine-induced contractions were inhibited by staurosporine but not by PD98059. We interpret these results to suggest that although ERK MAP kinases catalyze caldesmon phosphorylation, a second staurosporine sensitive kinase is also important in caldesmon phosphorylation and it is this pathway that may be more important in contractile regulation.     

Signaling pathway for nitric oxide generation with simulated ischemia in flow-adapted endothelial cells

Ischemia in the intact ventilated lung (oxygenated ischemia) leads to endothelial generation of reactive oxygen species (ROS) and nitric oxide (NO). This study investigated the signaling pathway for NO generation with oxygenated ischemia in bovine pulmonary artery endothelial cells (BPAEC) that were flow adapted in vitro. BPAECs were cultured in an artificial capillary system and subjected to abrupt cessation of flow (ischemia) under conditions where cellular oxygenation was maintained. Immunoblotting and dichlorofluorescein/triazolofluorescein fluorescence were used to assess extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation and ROS/NO generation, respectively. ERK1/2 phosphorylation significantly increased during ischemia, whereas total ERK1/2 did not change. ERK1/2 phosphorylation was suppressed by an inhibitor of tyrosine phosphorylation (genestein), cholesterol-binding reagents (filipin or cyclodextrin), or inhibitors of ROS (diphenyleneiodonium, N-acetylcysteine, or catalase), suggesting a role for both membrane cholesterol and ROS in ERK1/2 activation. Ischemia resulted in a 1.8-fold increase in NO generation that was suppressed by inhibitors of ERK1/2 activation (PD-98059 or U-0126). A calmodulin inhibitor (calmidizolium) or removal of Ca2+ from the medium also blocked NO generation, indicating that endothelial NO synthase (eNOS) is the activated isoform. These results indicate ischemia induces NO generation (possibly through a membrane cholesterol-sensitive flow sensor), the ERK1/2 cascade mediates signaling from the sensor to eNOS, and ROS are required for ERK activation.     

MEK/MAPK as a signaling element in ATP control of endothelial myosin light chain

Phosphorylation of endothelial myosin light chains (MLC) is a key mechanism in control of endothelial contractile machinery. Extracellular ATP influences endothelial MLC phosphorylation by either activation of Ca²⁺-dependent MLC kinase or Ca²⁺-independent MLC phosphatase. Here, the role of the MEK/MAPK pathway in this signaling was investigated in porcine aortic endothelial cells. Phosphorylation of ERK2 and phosphorylation of MLC were analyzed in cultured aortic endothelial cells. ATP (10 µM) increased ERK2 phosphorylation from basal 17 ± 3 to 53 ± 4%, an effect suppressed in the presence of the MEK inhibitors PD-98059 (20 µM) or U0126 (10 µM). Phosphorylation of ERK2 was not dependent on the ATP-induced cytosolic Ca²⁺ rise, because it was unaltered when this was suppressed by the Ca²⁺ chelator BAPTA (10 µM) or xestospongin C (3 µM), an inhibitor of the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ release mechanism of the endoplasmic reticulum. Phosphorylation of ERK2 was neither induced by the adenosine analog 5'-(N-ethylcarboxamido)adenosine (1 µM) nor inhibited in the presence of the adenosine receptor antagonist 8-phenyltheophylline (10 µM). ATP increased MLC kinase activity, and this was blocked in presence of PD-98059. ATP also increased MLC phosphatase activity, which was not inhibited by PD-98059. The MEK/MAPK pathway is a Ca²⁺-independent part of ATP signaling toward MLC kinase but not of ATP signaling toward MLC phosphatase. mitogen-activated protein kinase; contractile machinery; myosin light chain kinase; myosin light chain phosphatase     

Effects of hypoxia on relationships between cytosolic and mitochondrial NAD(P)H redox and superoxide generation in coronary arterial smooth muscle

Since controversy exists on how hypoxia influences vascular reactive oxygen species (ROS) generation, and our previous work provided evidence that it relaxes endothelium-denuded bovine coronary arteries (BCA) in a ROS-independent manner by promoting cytosolic NADPH oxidation, we examined how hypoxia alters relationships between cytosolic and mitochondrial NAD(P)H redox and superoxide generation in BCA. Methods were developed to image and interpret the effects of hypoxia on NAD(P)H redox based on its autofluorescence in the cytosolic, mitochondrial, and nuclear regions of smooth muscle cells isolated from BCA. Aspects of anaerobic glycolysis and cytosolic NADH redox in BCA were assessed from measurements of lactate and pyruvate. Imaging changes in mitosox and dehydroethidium fluorescence were used to detect changes in mitochondrial and cytosolic-nuclear superoxide, respectively. Hypoxia appeared to increase mitochondrial and decrease cytosolic-nuclear superoxide under conditions associated with increased cytosolic NADH (lactate/pyruvate), mitochondrial NAD(P)H, and hyperpolarization of mitochondria detected by tetramethylrhodamine methyl-ester perchlorate fluorescence. Rotenone appeared to increase mitochondrial NAD(P)H and superoxide, suggesting hypoxia could increase superoxide generation by complex I. However, hypoxia decreased mitochondrial superoxide in the presence of contraction to 30 mM KCl, associated with decreased mitochondrial NAD(P)H. Thus, while hypoxia augments NAD(P)H redox associated with increased mitochondrial superoxide, contraction with KCl reverses these effects of hypoxia on mitochondrial superoxide, suggesting mitochondrial ROS increases do not mediate hypoxic relaxation in BCA. Since hypoxia lowers pyruvate, and pyruvate inhibits hypoxia-elicited relaxation and NADPH oxidation in BCA, mitochondrial control of pyruvate metabolism associated with cytosolic NADPH redox regulation could contribute to sensing hypoxia.     

5,6-EET-induced contraction of intralobar pulmonary arteries depends on the activation of Rho-kinase.

The mechanism mediating epoxyeicosatrienoic acid (EET)-induced contraction of intralobar pulmonary arteries (PA) is currently unknown. EET-induced contraction of PA has been reported to require intact endothelium and activation of the thromboxane/endoperoxide (TP) receptor. Because TP receptor occupation with the thromboxane mimetic U-46619 contracts pulmonary artery via Rho-kinase activation, we examined the hypothesis that 5,6-EET-induced contraction of intralobar rabbit pulmonary arteries is mediated by a Rho-kinase-dependent signaling pathway. In isolated rings of second-order intralobar PA (1-2 mm OD) at basal tension, 5,6-EET (0.3-10 microM) induced increases in active tension that were inhibited by Y-27632 (1 microM) and HA-1077 (10 microM), selective inhibitors of Rho-kinase activity. In PA in which smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)) was increased with KCl (25 mM) to produce a submaximal contraction, 5,6-EET (1 microM) induced a contraction that was 7.0 +/- 1.6 times greater than without KCl. 5,6-EET (10 microM) also contracted beta-escin permeabilized PA in which [Ca(2+)](i) was clamped at a concentration resulting in a submaximal contraction. Y-27632 inhibited the 5,6-EET-induced contraction in permeabilized PA. 5,6-EET (10 microM) increased phosphorylation of myosin light chain (MLC), increasing the ratio of phosphorylated MLC/total MLC from 0.10 +/- 0.03 to 0.30 +/- 0.02. Y-27632 prevented this increase in MLC phosphorylation. These data suggest that 5,6-EET induces contraction in intralobar PA by increasing Rho-kinase activity, phosphorylating MLC, and increasing the Ca(2+) sensitivity of the contractile apparatus.