Abstract The generation of oxygen-derived free radicals has been implicated in the disordered vascular regulation of inflammation and reperfusion. In the vasculature, oxygen-derived free radicals are vasodilatory. The mechanisms underlying this effect remain unclear. To examine the cellular processes involved, we studied the effects of hydrogen peroxide (H2O2) on adenylyl cyclase activity in A10 cells, a murine vascular smooth muscle cell line. Pretreatment with H2O2 caused a dose-dependent enhancement of forskolin-stimulated adenylyl cyclase activity (ED50, 44 μmol/L to a maximum of 166% of control activity; n=4). This enhancement was attenuated by iron chelation with deferoxamine and by the intracellular hydroxyl scavenger dimethylthiourea and mimicked by preincubation with purine/xanthine oxidase either alone or in the presence of superoxide dismutase. The effects of H2O2 were completely blocked by the tyrosine kinase inhibitors genistein and tyrphostin A9 but not by its inactive analogue tyrphostin A1 (H2O2 alone, 149±13%; H2O2+tyrphostin A9, 100±9%; H2O2+tyrphostin A1, 171±21%; n=4). H2O2 comparably enhanced adenylyl cyclase activity stimulated by isoproterenol (166±17% of control, n=5) and sodium fluoride (177±18% of control, n=5). Thus oxygen-derived free radicals enhance adenylyl cyclase activation, probably via tyrosine kinase–mediated effects on the catalytic subunit of adenylyl cyclase. Sensitization of adenylyl cyclase activation may be an important mechanism by which free radicals modulate hormone-mediated vasodilation.
The generation of oxygen-derived free radicals has been implicated in several physiological and pathophysiological mechanisms of cardiovascular regulation. In the vasculature, a physiological role for oxygen-derived free radicals (including the superoxide anion, hydrogen peroxide [H2O2], and hydroxyl radicals) in local control of blood flow has been suggested previously.1 2 Further, free radical generation in the settings of inflammation, reperfusion after ischemic injury, and hypertension has been suggested to be an important factor in the disordered vascular regulation that occurs in these conditions.3 4 5 The formation of reduced oxygen intermediates occurs either by a pathway of superoxide anion→→H2O2→→hydroxyl radical→→water or alternatively via a combination of oxygen with organic compounds having two paired electrons.6 Of note, nitric oxide has been demonstrated to combine with oxygen to generate peroxynitrite, which decays homolytically to form the hydroxyl radical and nitrogen dioxide. This pathway may be of importance in oxygen-derived free radical generation in the setting of increased constitutive or inducible nitric oxide synthase activity.7
Both in vivo and in vitro studies have suggested that the primary direct effect of H2O2 and hydroxyl radicals is to mediate vasodilation1 2 3 4 8 (although endothelium-dependent vasoconstrictor effects have also been reported2 9 ). The mechanisms underlying these effects are unclear. Activation of second messenger systems, specifically guanylyl cyclase and adenylyl cyclase, represent two of the most important hormone-mediated signaling systems involved in vascular relaxation, A potential role of oxygen-derived free radicals in modulating guanylyl cyclase activation has been suggested previously.8 However, the effects of oxygen-derived free radicals on adenylyl cyclase activation remain to be established.
We were especially interested in studying the effects of the oxidant stressor H2O2. In other cell systems, H2O2 has been shown to be “insulinomimetic,” increasing insulin receptor kinase activity as well as insulin-mediated lipogenesis and protein synthesis.10 11 12 In previous studies, we have demonstrated that insulin sensitizes lymphocyte adenylyl cyclase activation.13 However, whether this effect on adenylyl cyclase is common to H2O2 and occurs in vascular smooth muscle cells was unknown.
On the basis of these uncertainties, the present studies were designed to assess the potential role of oxygen-derived free radicals in adenylyl cyclase activation in A10 cells. This rat embryonal thoracic aortic cell line demonstrates characteristics similar to vascular smooth muscle cells14 and has been a useful model with which to study vascular cellular processes.15 16 The data to be presented demonstrate that oxygen-derived free radicals enhance adenylyl cyclase activation probably via sensitization of the catalytic moiety of adenylyl cyclase and that this effect is mediated in part by hydroxyl radicals and acts via a tyrosine kinase–dependent mechanism.
Materials and Methods
A10 cells (obtained from American Type Culture Collection) were cultured in 48- and 24-well plates in DMEM with 5% fetal calf serum. Assays were performed on cells in stationary growth phase cultures and at least 4 days after the addition of fresh medium.
Assessment of Adenylyl Cyclase Activity
Assays of adenylyl cyclase activity were performed in permeabilized cells according to our previously described methods,13 which were modified to accommodate an adherent cell preparation (as opposed to our prior cell suspension preparations), and preparations were placed in multiwell culture plates. Medium was aspirated from the multiwell culture plates, and cells were washed in HBSS (pH 7.4 at 4°C) with 33 mmol/L HEPES, 0.5 mmol/L EDTA, and 1 mmol/L magnesium sulfate (buffer A). Cells were permeabilized with the addition of digitonin (10 μg/mL) in buffer A and incubated for 25 minutes at 4°C. Cells then were washed in buffer A without digitonin, followed by washing in HBSS (pH 7.4 at 4°C) with 33 mmol/L HEPES, 1.25 mmol/L EDTA, and 5 mmol/L MgSO4 (buffer B), and adenylyl cyclase activity was assessed by the conversion of [α-32P]ATP to [32P]cAMP.13 Briefly, permeabilized cells were incubated in a final volume of 200 μL with 1 μCi [α-32P]ATP (Amersham), 0.3 mmol/L ATP, 2 mmol/L magnesium sulfate, 0.1 mmol/L cAMP, 5 mmol/L phosphoenolpyruvate, 40 mg/mL pyruvate kinase, 20 mg/mL myokinase, 1 μg/mL BSA, 0.5 mmol/L ascorbic acid, and 0.5 mmol/L EDTA. Cells were incubated for 20 minutes at 37°C. Incubations were terminated by the addition of 1 mL of a solution containing 100 μg ATP, 50 μg cAMP, and 15 000 cpm [3H]cAMP (New England Nuclear). cAMP was isolated in the supernatant by sequential Dowex and alumina chromatography and corrected for recovery with [3H]cAMP as the internal standard. Initial studies demonstrated that adenylyl cyclase activity was linear with time and cell concentration over the range studied. Protein concentration in permeabilized cells was determined by the method of Bradford.17
Assessment of cAMP-Dependent Protein Kinase Activation
Assays of cAMP-dependent protein kinase activity were performed in permeabilized cells according to modifications of our previous methods using suspended-cell preparations.13 A10 cells were permeabilized as described above. Permeabilized cells in buffer B were incubated for 20 minutes at 30°C with 1 mmol/L Kemptide (Sigma), 0.5 mmol/L isobutyl methylxanthine, 1 μg/mL BSA, 0.5 mmol/L ascorbic acid, 0.8 mmol/L ATP, and 1 to 2 μCi [γ-32P]ATP in a final volume of 125 μL. Reactions were terminated by spotting aliquots (80 μL) on 2×3-cm phosphocellulose strips (Whatman P-81) and immersing them in 75 mmol/L phosphoric acid. The strips were swirled gently for 2 minutes, the phosphoric acid was decanted, and the strips were washed five more times as described above. Radioactivity was measured by liquid scintillation counting (Beckman LS 6000). Background was determined by blanks incubated in the absence of Kemptide, cells, or [γ-32P]ATP alone and generally accounted for <15% of basal activity. Protein kinase activity was linear with time up to at least 30 minutes.
Assessment of Toxin-Mediated G-Protein Labeling
The ADP ribosylation of G proteins by PT was carried out according to the method of Kopf and Woolkalis18 and was modified to accommodate for an adherent cell population and performed in multiwell culture plates. PT (100 μg/mL) was preactivated in a solution of 50 mmol/L HEPES, pH 8.0, 1 mg/mL BSA, 0.125% SDS, and 20 mmol/L DTT at 30°C for 30 minutes. Preactivated PT incubated with [32P]NAD (50 to 100 μCi/mL), 2.5 μmol/L NAD, and 500 μmol/L β-NADP was added to an assay mixture containing 1 mmol/L EDTA and 10 mmol/L thymidine. Addition of the preactivation mixture (with SDS) with cells resulted in cell lysis. Reaction mixtures incubated at 30°C for 30 minutes were terminated by the addition of 1 mL ice-cold buffer solution consisting of 5 mmol/L Tris-HCl and 3 mmol/L EDTA, pH 7.6. Lysates were recovered by centrifugation at 12 000g for 5 minutes. The pellet was washed with the same buffer and centrifuged again.
The ADP ribosylation of G proteins by CT was carried out according to the method of Gill and Woolkalis19 in multiwell cell culture plates and modified for an adherent cell population. CT (100 μg/mL) was preactivated in a solution of 50 mmol/L HEPES, pH 8.0, 1 mg/mL BSA, 0.125% SDS, and 20 mmol/L DTT at 30°C for 30 minutes. Preactivated CT incubated with [32P]NAD (50 to 100 μCi/mL), 2.5 μmol/L NAD, 500 μmol/L β-NADP, 100 μmol/L GTP, 1 mmol/L EDTA, and 10 mmol/L thymidine was added in a volume of 120 μL to intact adherent cells. Reaction mixtures were terminated, and lysates were obtained as described above.
SDS-PAGE was performed by using the procedure as described by Laemmli.20 Lysates (20 to 30 μg protein; equivalent amounts of protein were matched in each pair of samples analyzed) were dissolved in 50 μL sample buffer containing 125 mmol/L Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 10% 2-mercaptoethanol, and 0.025% bromophenol blue and boiled for 5 minutes before application to the gel. Protein molecular weight markers were dissolved in the same buffer. A 12% polyacrylamide running gel with a 4% stacking gel was used for all studies (model SE-400 gel apparatus, Hoefer). Electrophoresis was performed at a fixed current of 10 mA per gel slab for 15 to 18 hours. Gels were stained with 2% Coomassie blue R-250, 50% methanol, and 10% acetic acid for 30 minutes, followed by rapid destaining in 50% methanol, 10% acetic acid, and 3% glycerol for 3 to 4 hours. The gels were then air-dried overnight and exposed to x-ray film for 2 to 5 days at −80°C (Kodak X-AR). G-protein labeling was quantified by laser densitometric assessment of toxin-specific labeling (LKB 2222-020, Pharmacia-LKB Biotechnology) in films demonstrating submaximal exposure of toxin-specific bands in both control and H2O2-treated samples. Initial studies demonstrated that these conditions resulted in maximal toxin-mediated labeling.
For two-group comparisons, the statistical significance of differences was determined by Student’s t test for paired or unpaired data (where appropriate). For multiple-group comparisons, repeated-measures ANOVA was performed, followed by Dunnett’s multiple comparison test (where appropriate). A value of P<.05 on a two-sided test was taken as a minimum level of significance. Dose-response curves were analyzed by computerized nonlinear sigmoid curve fitting of the data (inplot 4, Graphpad Software).
H2O2-Mediated Enhancement of Adenylyl Cyclase Activation
Adenylyl cyclase activity in permeabilized A10 cells was stimulated above basal activity (6±1 pmol · min−1 · mg protein−1) by isoproterenol (100 μmol/L) to 55±3 pmol · min−1 · mg protein−1, by sodium fluoride (NaF, 10 mmol/L) to 112±11 pmol · min−1 · mg protein−1, and by forskolin (100 μmol/L) to 582±44 pmol · min−1 · mg protein−1 (n=5). To assess the effects of H2O2 and H2O2-derived free radicals on adenylyl cyclase activation, A10 cells resuspended in DMEM without fetal calf serum [with Fe(NO3)3, 250 nmol/L] were incubated in the presence and absence of H2O2 for 30 minutes. Subsequently, cells were permeabilized, and assays of adenylyl cyclase activity and cAMP-dependent protein kinase activity were performed as described above. Preincubation with H2O2 (100 μmol/L for 30 minutes) resulted in enhancement of stimulated adenylyl cyclase activity, ie, enhancement in response to a receptor-specific stimulator (isoproterenol, 100 μmol/L), a G-protein–specific stimulator (NaF, 10 mmol/L), and a catalytic subunit–selective stimulator (forskolin, 100 μmol/L) (Fig 1⇓). H2O2 comparably enhanced adenylyl cyclase activity stimulated by the nonhydrolyzable GTP analogue Gpp[NH]p (20 μmol/L, 154±10% of control, n=4). H2O2 mediated comparable enhancement of forskolin-stimulated adenylyl cyclase activity in cells that were serum-starved for 48 hours before H2O2 exposure (data not shown). H2O2 demonstrated a dose-dependent enhancement of forskolin-stimulated adenylyl cyclase activity with a ED50 of 44 μmol/L and a maximum to 166% of control forskolin-stimulated activity (n=4, Fig 2⇓). The effect of H2O2 to enhance adenylyl cyclase activity was linearly correlated with duration of exposure (r=.28, P=.025, Fig 3⇓).
Selective stimulation of the adenylyl cyclase catalytic subunit was also evaluated in the presence of 100 μmol/L forskolin plus 10 mmol/L MnCl2 in the absence of GTP and Mg2+ to prevent Gs activity.21 A10 cells incubated in the presence of H2O2 for 30 minutes were permeabilized, and adenylyl cyclase activity was assessed. H2O2 mediated a significant enhancement of forskolin+MnCl2–stimulated adenylyl cyclase activity (186±22% of control, n=3).
To determine whether enhancement of adenylyl cyclase responsiveness represented a membrane-delimited effect (as would be expected if it acted by direct oxidation of cysteine residues on adenylyl cyclase versus an effect dependent on an intact cell system), A10 cells were permeabilized, scraped from the culture dish with a rubber policeman, resuspended in buffer B with PMSF (10 μmol/L), and homogenized in a Potter-Elvehjem apparatus, at which time no intact cells were seen on light microscopy. Membrane preparations were centrifuged at 20 000g for 20 minutes and resuspended in a minimal volume of buffer B with PMSF (10 μmol/L). Aliquots were then incubated for 30 minutes with or without H2O2+250 nmol/L Fe(NO3)3 at 37°C for 30 minutes, followed by the addition of assay mixture for assessment of adenylyl cyclase activity as described above.
NaF/AlCl3 (20 mmol/L:20 μmol/L)–stimulated adenylyl cyclase activity assessed in membrane preparations made from cells pretreated with H2O2 was enhanced (Table 1⇓). However, H2O2 treatment of membrane preparations resulted in no enhancement of adenylyl cyclase activity (Table 1⇓).
The functional effects of H2O2-mediated enhancement of adenylyl cyclase activity were still evident at the level of A-kinase. A-kinase activity in permeabilized A10 cells was stimulated above basal activity (208±89 pmol phosphoprotein · min−1 · mg protein−1) by forskolin (1 μmol/L) to 501±28 pmol phosphoprotein · min−1 · mg protein−1, and by cAMP (100 μmol/L) to 4625±385 pmol phosphoprotein · min−1 · mg protein−1. H2O2 (100 μmol/L) pretreatment was associated with a significant enhancement of forskolin-stimulated A-kinase activity (145±8% of control, n=3) without significant alteration in cAMP-stimulated A-kinase activation (101±16%).
H2O2 may have direct effects on vascular cells. Alternatively H2O2 might act via formation of hydroxyl radicals as generated via the Haber-Weiss and/or Fenton reactions. To generate oxygen-derived hydroxyl radicals via the Fenton reaction, reduced iron (or other metal) is required as a cofactor. To examine the importance of iron (contained in the DMEM) in the H2O2-mediated enhancement of adenylyl cyclase activity, the effect of the iron chelator deferoxamine (10 mmol/L) was examined. Preincubation with deferoxamine was not associated with any significant enhancement in adenylyl cyclase activation in the absence of H2O2 (89±10% of control, n=8). However, deferoxamine treatment significantly attenuated the effect of H2O2 to enhance adenylyl cyclase activation (Fig 4⇓). To further examine this mechanism of effect, cells were incubated with H2O2 in the presence or absence of excess catalase (760 U/mL), which catalyzes the conversion of H2O2 to water. The effect of H2O2 to enhance NaF-stimulated adenylyl cyclase activity was blunted almost completely by coincubation with catalase (H2O2, 185±28% of control; H2O2+catalase, 120±6% of control; n=3).
To determine whether hydroxyl radicals acted at extracellular or intracellular sites, the effect of H2O2 was examined in the presence of mannitol (30 mmol/L), an extracellular hydroxyl radical scavenger, or DMTU (10 mmol/L), an intracellular hydroxyl radical scavenger, which were preincubated with cells for 15 minutes before the addition of H2O2. Neither DMTU nor mannitol significantly altered adenylyl cyclase stimulation in the absence of H2O2. However, DMTU but not mannitol significantly attenuated the H2O2-mediated enhancement of adenylyl cyclase activation (Fig 5⇓).
Effects of Xanthine Oxidase/Purine on Adenylyl Cyclase Activation
As an alternative approach to generating oxygen-derived free radicals, cells were incubated with xanthine oxidase (10 mU/mL) and purine (1.67 mmol/L) for 30 minutes at 37°C. Cells were permeabilized, and adenylyl cyclase activity was assessed as described above. Xanthine oxidase and purine pretreatment increased forskolin-stimulated adenylyl cyclase activity to 127±5% of control (P<.05, n=4, Fig 6⇓). Coincubation with superoxide dismutase (600 U/mL), which catalyzes the conversion of superoxide anions to H2O2, resulted in a comparable enhancement of adenylyl cyclase activation (124±7% of control, P<.05 versus control, n=4). In contrast, preincubation with both catalase (760 U/mL) and superoxide dismutase almost entirely blocked the enhancement of adenylyl cyclase activation mediated by xanthine oxidase and purine (108±6% of control). Further, pretreatment with deferoxamine (10 mmol/L) completely blocked the xanthine oxidase/purine-mediated enhancement of adenylyl cyclase activation (Fig 7⇓).
Effect of Protein Tyrosine Kinase Inhibitors on H2O2-Mediated Enhancement of Adenylyl Cyclase Activation
As noted above, previous studies have suggested that the effects of H2O2 in other systems were mediated by tyrosine kinase–dependent protein phosphorylation. To examine the potential role of this mechanism in the H2O2-mediated enhancement of adenylyl cyclase activity, cells exposed to H2O2 were incubated in the presence or absence of tyrphostin A9 (40 μmol/L), a potent inhibitor of protein tyrosine kinase, or tyrphostin A1 (40 μmol/L), an inactive analogue of tyrphostin A9. In the absence of H2O2, pretreatment of cells with either tyrphostin A1 or A9 was not associated with any significant alteration in NaF-stimulated adenylyl cyclase activation (Fig 8⇓). However, tyrphostin A9 pretreatment completely blunted the effect of H2O2 on adenylyl cyclase activation. In contrast, tyrphostin A1 was ineffective in blunting the H2O2-mediated effect (Fig 8⇓). Genistein (100 μmol/L), another inhibitor of tyrosine kinase, comparably inhibited the H2O2-mediated enhancement of adenylyl cyclase activity (Table 2⇓). Tyrphostin B46 comparably blunted the H2O2-mediated effect (data not shown).
Several tyrosine kinase–linked receptor systems mediate their intracellular effects by activation of phospholipase C and subsequently C-kinase (PKC).22 Further, PKC-mediated phosphorylation of mammalian adenylyl cyclase isoforms has been described previously.23 24 To assess the role of C-kinase activity in the H2O2-mediated enhancement of adenylyl cyclase activity, the effect of the selective C-kinase inhibitor BIM (500 nmol/L, a concentration associated with maximal inhibition of PKC) was examined. Preincubation with BIM at 37°C for 30 minutes had no effect on forskolin-stimulated adenylyl cyclase activity in the absence of H2O2 (100±2% of control, n=3). Further BIM treatment did not attenuate the effect of H2O2 on adenylyl cyclase activity (Fig 9⇓).
Effects of H2O2 on G-Protein Labeling
H2O2 mediated comparable enhancement of adenylyl cyclase activity in response to various stimulators. That is, adenylyl cyclase activation levels in response to a receptor-specific stimulator, a G-protein–specific stimulator, and an enzyme-specific stimulator (forskolin/Mn2+) all showed comparable enhancement. This suggests that H2O2 exerts its effect at the level of the catalytic subunit. However, we examined the potential effect of H2O2 on G proteins for two reasons: (1) Forskolin is not a selective stimulator of catalytic adenylyl cyclase function.25 26 27 28 (2) Tyrosine kinase regulation of G-protein function (including alterations in toxin-mediated [32P]ADP ribosylation of G-protein α-subunits) has been described previously.29 30 31 32 Hence, we assessed G proteins by toxin-mediated ADP ribosylation in the presence or absence of H2O2.
PT-specific [32P]NAD labeling identified an A10 vascular cell protein with a molecular mass of 41 kD (Fig 10⇓), consistent with labeling of the α-subunit of the guanine nucleotide regulatory protein of inhibition (Gi) and/or Go. CT-specific [32P]NAD labeling identified two A10 vascular cell proteins with molecular masses of 45 and 52 kD (Fig 11⇓), consistent with labeling of the long and short forms of the α-subunit of the guanine nucleotide regulatory protein of stimulation (Gs). Pretreatment of intact cells with H2O2 at 30°C for 30 minutes resulted in no alterations in the extent of either CT-mediated labeling (101±6% of control) or PT-mediated labeling (93±4% of control) (Fig 12⇓).
H2O2 and oxygen-derived free radicals modulate vasodilator mechanisms.1 2 3 4 5 6 9 The present studies indicate that H2O2 enhances adenylyl cyclase activation and that the effect is dependent (in part) on the presence of iron and is blunted by agents that act to inhibit tyrosine kinase activity.
Our data suggest that the oxygen-derived species mediating the enhancement of adenylyl cyclase activation is either H2O2 itself or the hydroxyl radical. Incubation of cells with xanthine oxidase and purine resulted in a qualitatively similar enhancement of adenylyl cyclase activation. The effect of purine and xanthine oxidase was not blocked by coincubation with superoxide dismutase (which catalyzes the conversion from superoxide anion to H2O2). This suggests that the generation of the superoxide anion is not involved in the mechanism of enhancement of adenylyl cyclase activation. However, pretreatment with either catalase (which catalyzes conversion of H2O2 to water) or with deferoxamine (which chelates divalent metal ions and inhibits hydroxyl radical formation via the Fenton reaction) did block the effect of both xanthine oxidase/purine and H2O2, implicating the production of H2O2 and/or hydroxyl radicals as the important reactive species. Further, the effect of H2O2 was blunted by the intracellular hydroxyl radical scavenger DMTU. The effect of H2O2 and/or hydroxyl radicals to enhance adenylyl cyclase activation is consistent with their previously described direct physiological effect of vasorelaxation (as described above).
The site of the effect of H2O2 and/or hydroxyl radicals on adenylyl cyclase activity appears to be at the level of the catalytic subunit. Adenylyl cyclase–linked transmembrane signaling systems consist of a ternary complex of receptor, guanine nucleotide regulatory protein (G protein), and catalytic subunit (adenylyl cyclase). To determine the site in the ternary complex primarily affected by H2O2, we examined the pattern of H2O2-mediated enhancement of adenylyl cyclase activity in response to receptor-based stimulation (via the β-adrenergic receptor agonist isoproterenol), G-protein–selective stimulators (via NaF and Gpp[NH]p), and catalytic subunit–selective stimulators (via forskolin/Mn2+). The comparable enhancement of adenylyl cyclase activation by H2O2 in response to all stimulators of adenylyl cyclase suggests that the major site of the H2O2-mediated effect is at the level of a catalytic subunit. However, additional effects at the level of the receptor and/or G proteins cannot be completely ruled out.
These studies suggest that the effects of H2O2 on the adenylyl cyclase complex are probably indirect. A direct effect of hydroxyl radicals (the oxidation of critical cysteine residues of type I adenylyl cyclase) recently has been reported.29 However, this latter effect was inhibitory (rather than the stimulatory effect seen in the present studies). Furthermore, the effect of H2O2 is unlikely to be due to direct oxidation of adenylyl cyclase, since it could not be seen when cell lysates were directly exposed to H2O2.
The results of our studies using the tyrosine kinase–selective inhibitors (tyrphostin A9, tyrphostin B46, and genistein) suggest that H2O2-mediated enhancement of adenylyl cyclase activation occurs via a tyrosine kinase–dependent pathway. As noted above, in several model systems, H2O2 has been shown to stimulate both receptor tyrosine kinase–mediated phosphorylation as well as more “downstream” insulin-mediated effects.10 11 12 Does the tyrosine kinase–dependent mechanism of H2O2-mediated enhancement of adenylyl cyclase activation help to localize which component of the ternary complex transmembrane signaling system is affected? As noted previously, tyrosine kinase–mediated regulation of G-protein function has been described and has been associated with both enhancement and depression of G-protein function.29 30 31 32 However, we were unable to identify an H2O2-mediated alteration in G proteins as assessed by toxin-mediated labeling. Notably, tyrosine kinase–dependent regulation of catalytic subunit function has not been previously demonstrated and thus would represent a novel mechanism of cross talk between tyrosine kinase–linked receptor systems and those signaling systems linked to activation of adenylyl cyclase. However, whether this effect is related to tyrosine phosphorylation of adenylyl cyclase or occurs via any one of the numerous effectors linked to an insulin-receptor signaling system (ras, phosphatidylinositol 3′-kinase, etc) remains to be determined. Notably, tyrosine kinase receptor activation has been reported to increase C-kinase activity via phospholipase C-γ (reviewed in Reference 2222 ). Further, C-kinase–mediated phosphorylation resulting in sensitization of some but not all isoforms of adenylyl cyclase has been reported previously.33 34 35 However, inhibition of C-kinase activity with BIM did not attenuate the effect of H2O2.
Whether these findings represent a correlate to the alterations in the adenylyl cyclase complex seen with ischemia/reperfusion is unclear. Both sensitization and desensitization of adenylyl cyclase activation have been reported with ischemia and reperfusion.36 37 38 Furthermore, these previous studies have focused on the ternary complex transmembrane signaling system in myocardial cells. The effects of oxidant stress associated with reperfusion on the regulation of adenylyl cyclase activation in vascular smooth muscle cells have not been studied.
In summary, the present study has demonstrated that oxygen-derived free radicals enhance adenylyl cyclase activation in vascular smooth muscle cells. It should be stressed that the significance of these findings regarding free radical regulation of vascular function in vivo has yet to be determined. However, the present study does suggest a novel mechanism by which oxidant stress might modulate vascular function both physiologically and pathophysiologically.
Selected Abbreviations and Acronyms
|A-kinase||=||cAMP-dependent protein kinase|
|BSA||=||bovine serum albumin|
|PKC||=||protein kinase C|
This study was supported by grants-in-aid from the Medical Research Council of Canada and Heart and Stroke Foundation of Ontario. Dr Feldman was supported by a Career Investigator Award from the Heart and Stroke Foundation of Ontario. The authors gratefully acknowledge the excellent technical support of W. DeYoung and J. Chorazyczewski.
- Received November 11, 1994.
- Accepted June 12, 1995.
- © 1995 American Heart Association, Inc.
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