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CARDIOVASCULAR
Department of Pharmacology, Monash University, Clayton, Victoria, Australia (C.P.J., G.R.D.); Department of Pharmacology, University of Melbourne, Victoria, Australia (C.G.S., A.A.M.); Howard Florey Institute, University of Melbourne, Victoria, Australia (T.T.D.); and Bernard O'Brien Institute of Microsurgery, University of Melbourne, Fitzroy, Victoria, Australia (G.J.D.)
Received October 8, 2005; accepted January 4, 2006.
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Abstract |
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,
-methylene-ATP. Importantly, ,-methylene-ATP had no effect on superoxide production induced by NADPH. In conclusion, these findings suggest little role for NADPH oxidase-derived superoxide in the contractile effects of NADPH in the mouse aorta. Rather, NADPH seems to act as an agonist at two distinct P2X receptor populations; one located on the endothelium and the other on smooth muscle layer, both of which ultimately lead to contraction.
; Madamanchi et al., 2005). NADPH oxidases generate superoxide by transferring electrons from the pyridine nucleotide NADPH (and possibly NADH; see Lassegue and Clempus, 2003) to molecular oxygen. These enzymes were first identified in neutrophils as the enzyme complex responsible for the respiratory burst; however, isoforms of NADPH oxidase have been identified recently in nonphagocytic cells, including all major cell types that make up the blood vessel wall (i.e., endothelial and smooth muscle cells and adventitial fibroblasts) (Lassegue and Clempus, 2003). At least three isoforms of NADPH oxidase have been identified in the vascular wall, all of which are built on the same basic molecular plan consisting of cytosolic organizer (p47phox or NoxO1) and activator (p67phox or NoxA1) subunits, a small G protein (Rac1 or Rac2), and a membrane-bound cytochrome-b558 reductase domain (Lassegue and Clempus, 2003). Cytochrome-b558 is actually a heterodimer made up of a small -subunit, p22phox, and a larger catalytic -subunit or Nox, the nature of which varies with different isoforms of the enzyme (Lassegue and Clempus, 2003). Within vascular cells, NADPH oxidase isoforms containing Nox1, Nox2 (gp91phox), and Nox4 have been identified (Bengtsson et al., 2003; Lassegue and Clempus, 2003).
In contrast to the NADPH oxidase expressed in phagocytes, which only becomes activated after exposure of cells to pathogens or proinflammatory mediators, a large pool of vascular NADPH oxidase is constitutively assembled (Li and Shah, 2002). This characteristic of the vascular enzyme means that addition of an excess of the pyridine nucleotide substrate, NADPH (or NADH) alone to vascular preparations is sufficient to increase superoxide production (Li and Shah, 2002). Interestingly, NADPH seems to be equally effective at driving superoxide production whether added to intact or permeabilized preparations (Pagano et al., 1995; Li and Shah, 2001; Souza et al., 2001; Ellmark et al., 2005), indicating either that extracellular NAD(P)H gains entry to the intracellular compartment by an undefined mechanism or that the NAD(P)H binding site of vascular NADPH oxidase is accessible from the extracellular surface of the plasma membrane.
ROS are known to elicit a range of effects on vascular tone ranging from H2O2-mediated opening of K+ channels and subsequent vasodilatation to vasoconstriction arising from superoxide-dependent inactivation of nitric oxide. Hence, a number of recent studies have examined the vasomotor effects of NADPH and NADH in vitro and in vivo to assess the role of NADPH oxidase in regulation of vascular tone (Souza et al., 2001; Didion and Faraci, 2002; Paravicini et al., 2004; Park et al., 2004). However, in addition to augmenting NADPH oxidase activity, pyridine nucleotides could conceivably modulate vascular tone via stimulation of P2 nucleotide receptors. Indeed, many blood vessels are known to express nucleotide receptors both on the endothelial and smooth muscle layers (Kunapuli and Daniel, 1998). Moreover, in nonvascular smooth muscle preparations, including the guinea pig taenia coli and rat anococcygeus muscle, responses to NADPH and NADH were due to activation of P2 and A1 receptors, respectively (Burnstock and Hoyle, 1985; Najbar et al., 1994). Of note, there were no qualitative differences between the oxidized [NAD(P)+; electron depleted] and reduced forms of each nucleotide in terms of their ability to modulate smooth muscle tone (Najbar et al., 1994), providing further evidence for a non-NADPH oxidase-dependent action.
In the present study, we examined the relative contributions of NADPH-dependent oxidases (e.g., NADPH oxidase and nitric-oxide synthase) and P2 nucleotide receptors to vascular responses (superoxide production and vasocontraction) elicited by NADPH in mouse isolated thoracic aortas for the following two reasons: 1) the mouse aorta is known to express a functional NADPH oxidase and addition of NADPH to intact rings has previously been shown to increase both superoxide production and vascular tone (Souza et al., 2001); and 2) P2 receptors were recently established to be expressed on both the endothelium and vascular smooth muscle cell layers of the mouse aorta, and these receptors could conceivably contribute to the established vasoconstrictor effects of NADPH in this tissue (Beny, 2004). Our findings confirm previous observations that NADPH increases superoxide production from NADPH oxidase in the mouse aorta. However, this action does not contribute to the vasoconstrictor effects of NADPH in this tissue. Rather, activation of P2X receptors on both the endothelium and smooth muscle layers is the predominant mechanism by which NADPH modulates aortic tone.
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Materials and Methods |
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Lucigenin-Enhanced Chemiluminescence. Following isolation, the thoracic aorta was immediately placed in ice-cold Krebs-HEPES buffer containing 99 mM NaCl, 4.7 mM KCl, 1.0 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 25 mM NaHCO3, 20 mM Na-HEPES, and 11 mM glucose, pH 7.4. Aortas were cleared of adherent fat and cut into 2-mm rings. In some rings, the endothelium was removed by gently abrading the luminal surface of the vessel with a stainless steel pin. Vascular superoxide production was measured by 5 µM lucigenin-enhanced chemiluminescence as described previously (Paravicini et al., 2002). In brief, aortic rings were preincubated for 45 min at 37°C in Krebs-HEPES buffer containing either no further additives or one or more of the following drugs: 3 mM diethyldithiocarbamic acid [DETCA; to inactivate endogenous superoxide dismutase (SOD)1 and SOD3 activity], 1 to 3000 µM NADPH, 100 µM NADH, 100 µM NADP+, 5 µM diphenyleneiodonium, 1 mM apocynin, 300 units/ml native Cu2+/Zn2+-(SOD1) and 60 units/ml native Mn2+-superoxide dismutase (SOD2), 30 µM M40403, 10 mM tiron, 100 µM NG-nitro-L-arginine methyl ester (L-NAME), 3 µM indomethacin, 100 µM allopurinol, 1 µM rotenone, and 10 µM ,-methylene-ATP. Aortic rings were then transferred into the wells of an opaque white 96-well plate, each of which contained 300 µl of a Krebs-HEPES-based assay solution consisting of 5 µM lucigenin as well as the appropriate substrate/inhibitor compound(s). The plate was then placed into a TopCount Microplate scintillation/luminescence counter (Packard, Boston, MA), and tissue-dependent photon emission per second per well was monitored over a 20-min period. At the completion of the assay, aortic rings were dried in a 60°C oven for 24 h, enabling superoxide production to be normalized to dry tissue weight.
Isometric Tension Assay. Abdominal aortas were placed into ice-cold Krebs-bicarbonate buffer [118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 25 mM NaHCO3, and 11 mM glucose (equilibrated with 95% O2, 5%CO2)] and cut into four 2-mm ring segments. Rings were then placed into the chambers of a myograph (model 610M; Danish Myo Technology A/S, Aarhus N, Denmark) with ice-cold Krebs-bicarbonate buffer and gradually warmed to 37°C before being mounted on two stainless steel wire calipers (100 µm in diameter), with one caliper connected to a force displacement transducer and the other caliper to a micrometer. Rings were then incubated under zero tension for 20 min, after which time the baseline tension was elevated to 5 mN by manually adjusting the micrometer. After a further 30 min, maximal contraction of the tissue (Umax) was determined using 0.3 µM U46619
[GenBank]
for 15 min. Aortic rings were then washed with fresh Krebs (three times) and allowed to return to baseline tension (
40 min). Some rings were then treated for 20 min with one of the following inhibitor drugs: 10 mM tiron, 5 µM diphenyleneiodonium, 100 µM L-NAME, 10 µM ,-methylene-ATP, 100 µM PPADS, or 10 µM NF023. Tissues were then precontracted to between 45 and 55% Umax by titrating the concentration of U46619
[GenBank]
in the organ chamber. Once a stable plateau of tone was attained, cumulative half-log molar concentrations of either NADPH, NADP+, or NADH (1–300 µM) were added. The experiment was completed by addition of 30 µM acetylcholine chloride (Ach) to confirm the presence or absence of a functional endothelium.
Drugs. Ach, allopurinol, ,-methylene-ATP lithium salt, NADP+, NADPH, NADH, Cu2+/Zn2+-superoxide dismutase from bovine erythrocytes (SOD1), U46619
[GenBank]
, 4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt (tiron), diphenyleneiodonium chloride, 4'-hydroxy-3'-methoxyacetophenone (apocynin), indomethacin, NF023, L-NAME, N,N'-dimethyl-9,9'-biacridinium dinitrate (lucigenin), sodium diethyldithiocarbamic acid trihydrate (DETCA), rotenone, and pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt (PPADS) were all purchased from Sigma-Aldrich (St. Louis, MO). M40403 [a manganese(II)-bis(cyclohexylpyridine)-substituted macrocyclic superoxide dismutase mimetic] was obtained from Metaphor Pharmaceuticals (St. Louis, MO).
Data Analysis. Superoxide production was expressed either as relative light units per second per milligram of dry tissue weight (Fig. 1A) or as a percentage of the median control value of each experimental group. Umax values are expressed in millinewtons, whereas changes in isometric tension to NADPH, NADP+, and NADH are expressed as a percentage of the U46619 [GenBank] -induced precontraction. All results are expressed as mean ± S.E.M. of experiments conducted on tissues taken from n animals. Concentration-dependent response curves for NADPH, NADP+, and NADH were computer fitted (GraphPad Prism version 4.00; GraphPad Software Inc., San Diego, CA) with a sigmoidal regression curve of the following equation: Y = Bottom + (Top – Bottom)/1 + 10(pEC50–X)nH, where X is the logarithm of the agonist concentration, Y is the response (i.e., superoxide production or vasocontraction), Bottom is the lower response plateau, Top is the upper response plateau, and pEC50 is the logarithm of the X value when the response is half-way between Bottom and Top. The variable nH controls the slope of the curve. Statistical comparisons were made using either Student's t test or one- or two-way analysis of variance with Tukey-Kramer's post hoc test. A value of P < 0.05 was considered statistically significant.
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50% by mechanical removal of the endothelium (Fig. 1C).
In isometric tension studies, NADPH caused concentration-dependent contractions in rings of aorta precontracted with U46619
[GenBank]
(Fig. 2, A and B). NADPH elicited a similar effect on vascular tone in rings of artery precontracted with phenylephrine but had no effect in rings maintained under resting tension (Supplemental Figure 2). Removal of the endothelium inhibited contractions to NADPH by 50% (Fig. 2B) and relaxations to Ach by 80% (Fig. 2C). Interestingly, tiron had no significant effect on contractions to NADPH in endothelium-intact tissues but markedly attenuated the response in endothelium-denuded tissues (Fig. 2A). Tiron had no effect on responses to Ach in endothelium-intact or -denuded tissues (Fig. 2B).
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), also had no significant effect on superoxide levels (Fig. 3A). Surprisingly, despite its inability to stimulate superoxide production, NADP+ was equally as effective as NADPH at eliciting contractions in precontracted mouse aortic rings (Fig. 3B). In contrast, NADH induced a small relaxation response (Fig. 3B).
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The above-mentioned findings indicate that the effect of NADPH on aortic tone may not always be related to its ability to augment superoxide production from NADPH-dependent vascular oxidases such as NADPH oxidase. To further explore this possibility, we compared the effects of known inhibitors of NADPH oxidase and other potential sources of vascular ROS, on superoxide production and contraction. NADPH-dependent superoxide production was significantly reduced after acute exposure of aortic rings to the flavin antagonist and nonselective NADPH oxidase inhibitor diphenyleneiodonium (P < 0.001; n = 6; Fig. 4A). Consistent with our previous observation in mouse cultured vascular smooth muscle cells (Ellmark et al., 2005), short-term incubation with apocynin failed to reduce NADPH-dependent superoxide production (Fig. 4A). In contrast, when the exposure time to apocynin was increased to 24 h, NADPH-stimulated superoxide production seemed to be markedly attenuated (Fig. 4B); however, this effect just failed to reach statistical significance because of the large variation in control responses to NADPH in aortic rings after 24 h of organoid culture. L-NAME, an inhibitor of nitric-oxide synthase, also seemed to reduce NADPH-dependent superoxide production by 40%, but again this just failed to reach significance, whereas inhibitors of cyclooxygenase (indomethacin), xanthine oxidase (allopurinol), and the mitochondrial electron transport chain (rotenone) had no effect (Fig. 4C). These results suggest that NADPH oxidase (and possibly endothelial nitric-oxide synthase) contribute to vascular superoxide production in response to NADPH. However, neither diphenyleneiodonium nor L-NAME had any effect on the contractile response to NADPH in isolated aortic rings (Fig. 5A), despite both treatments inhibiting endothelium-dependent relaxations to Ach (Fig. 5B).
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Vasocontractions to NADPH Are Mediated by P2X Receptors. NADPH has previously been described as an agonist of P2 receptors (Burnstock and Hoyle, 1985; Najbar et al., 1994). Therefore, we examined the influence of ,-methylene-ATP (a P2X-desensitizing agent) on NADPH-induced vasocontractions in mouse isolated aorta. ,-methylene-ATP caused large contractions, which were similar in magnitude in endothelium-intact and -denuded rings of aorta. In both cases, responses to ,-methylene-ATP spontaneously returned to baseline levels of tension after 3 min, presumably reflecting desensitization of the P2X effector pathways that lead to vasocontraction in this tissue. Pretreatment with ,-methylene-ATP had no effect on the concentration of U46619
[GenBank]
required to precontract tissues to 50% Umax but it markedly attenuated responses to NADPH in both endothelium-intact and -denuded tissues (Fig. 6A). In contrast, ,-methylene-ATP had no effect on NADPH-induced superoxide production in mouse aortic rings (Fig. 6B), further highlighting the fact that NADPH induces vasocontraction and superoxide production via different mechanisms.
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To confirm the involvement of P2X receptors in vasocontractions to NADPH, we examined the effects of PPADS, a broad-selectivity P2X receptor antagonist, and NF023, which is a relatively selective P2X1 antagonist. Vasocontractions to NADPH in both endothelium-denuded and -intact tissues were virtually abolished in the presence of PPADS (Fig. 7A). NF023 also inhibited vasocontractions to NADPH. In endothelium-intact tissues, NF023 suppressed responses to NADPH by 50%. In endothelium-denuded tissues, NF023 seemed to cause an 10-fold rightward displacement of responses to NADPH, which would fit with its reported action as a competitive antagonist of P2X1 receptors.
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; Didion and Faraci, 2002; Paravicini et al., 2004; Park et al., 2004). However, here we have shown that contractile responses to NADPH in the mouse isolated aorta are not dependent on NADPH oxidase activity. Thus, although exogenous NADPH was highly effective at augmenting NADPH oxidase activity, its contractile effects were insensitive to diphenyleneiodonium and L-NAME, excluding any role for NADPH oxidase and superoxide-dependent inactivation of NO in the vasomotor response. Rather, vasocontractions to NADPH were mediated by activation of two distinct populations of P2X receptors, one population located on the endothelium and the other population on the smooth muscle layer.
In this study, as in previous studies by our group (Paravicini et al., 2002, 2004) and others (Pagano et al., 1995), exogenous NADPH stimulated superoxide production in intact vascular ring segments. Superoxide production was virtually abolished by diphenyleneiodonium and was markedly attenuated by apocynin, suggesting that NADPH oxidase is primarily responsible for superoxide production in response to NADPH in mouse isolated aortic segments. These findings obtained using the lucigenin technique are thus consistent with those of Souza et al. (2001), who, using electron paramagnetic spin resonance, similarly showed that extracellular NADPH stimulates superoxide production in intact aortic segments. Furthermore, these authors showed that superoxide production in response to NADPH was similar in aortas from wild-type and Nox2-knockout mice, implicating a nonphagocytic isoform of NADPH oxidase in the response (Souza et al., 2001). Although it remains to be determined which isoform(s) is involved in supporting NADPH-dependent superoxide production in whole ring segments, we have recently demonstrated a major role for a Nox4-containing NADPH oxidase in NADPH-dependent superoxide production in mouse cultured aortic smooth muscle cells (Ellmark et al., 2005). This is further supported by our observation that Nox4 is by far the most abundantly expressed NADPH oxidase isoform in the mouse aorta (Bengtsson et al., 2003).
In addition to elevating superoxide production, exogenous NADPH also caused concentration-dependent contractions in mouse aortic rings, which were partially dependent on the presence of a functional endothelium. However, our data indicate that NADPH did not mediate contraction via its ability to act as a reducing equivalent for NADPH oxidase-dependent superoxide generation and subsequent inactivation of NO. First, removal of the endothelium inhibited contractions to NADPH yet augmented superoxide production, this latter effect suggesting that endothelial cells exert a tonic suppressive effect on NADPH oxidase activity. Second, NADP+, which lacks a donatable hydride ion, was equally as effective as NADPH at causing contraction. Third, diphenyleneiodonium, which abolished NADPH oxidase-dependent superoxide production in mouse aortic rings, had no effect on NADPH-induced contractions. Finally, contractions to NADPH were not affected by the NOS inhibitor L-NAME, indicating that they were independent of NO. Rather, both the endothelium-dependent and -independent components of the contractile response to NADPH were virtually abolished by the P2X receptor desensitizing agent ,-methylene-ATP and were markedly inhibited by the P2X antagonists PPADS and NF023, implicating a role for P2X receptors in the response.
Although NADPH has previously been shown to display P2Y-like effects in the rat isolated mesenteric arterial bed (Ralevic and Burnstock, 1996), to our knowledge this is the first report that NADP(H) may act as a direct agonist at P2X receptors. P2X receptors are members of the ligand-gated ion channel superfamily and are characterized by their inwardly rectified permeability to Ca2+ and other cations in response to ATP and related nucleotides. Within the vasculature, P2X1 seems to be the predominant P2X subtype expressed in smooth muscle cells (Kunapuli and Daniel, 1998; North, 2002). The P2X receptor profile of endothelial cells is less well characterized; however, the predominant receptor subtypes on this cell type probably include P2X1 (Harrington and Mitchell, 2004), P2X4 (Glass et al., 2002), P2X5 (Schwiebert et al., 2002), and P2X7 (Ramirez and Kunze, 2002) receptors. Moreover, there is some evidence for regional variation (e.g., artery versus vein) in endothelial P2X receptor expression throughout the vasculature (Ray et al., 2002). Our finding that NF023, at a concentration that selectively targets P2X1 receptors (North, 2002), caused an 3-fold rightward shift of the contraction response in endothelium-denuded rings certainly supports the idea that P2X1 are involved in the direct smooth muscle effects of NADPH. Responses to NADPH in rings with an intact endothelium were also partially impaired by NF023; however, given that we were unable to examine the endothelium-dependent component of the response in isolation from the direct smooth muscle effect of NADPH, it is not possible to conclude whether NF023 also has an effect at the endothelial level. Nevertheless, we did find that PPADS and ,-methylene-ATP virtually abolished both the endothelium-dependent and -independent components of the response to NADPH. ,-methylene-ATP is selective for P2X1 and P2X3 receptor subtypes (North, 2002). Given that there is little evidence for P2X3 receptors on endothelial cells, these findings with ,-methylene-ATP probably indicate that the endothelial receptor involved is also of the P2X1 subtype.
Although it is not surprising that activation of VSMC P2X receptors would lead to contractions via an increase in VSMC Ca2+, it is somewhat surprising that stimulation of endothelial P2X receptors is also linked to contraction, since rises in endothelial cell Ca2+ are normally associated with release of endothelium-derived relaxing factors such as NO (and in certain vascular beds prostacyclin and endothelium-derived hyperpolarizing factor). Previous studies have demonstrated that stimulation of endothelial P2Y receptors results in contraction via the release of thromboxane (Shirahase et al., 1991). However, the release of a vasoconstrictor prostanoid does not explain the endothelium-dependent vasocontraction effects to NADPH observed here because the cyclooxygenase inhibitor indomethacin had no effect (Supplemental Fig. 3). Hence, the mechanism by which P2X receptor stimulation leads to endothelium-dependent contraction remains to be elucidated.
In conclusion, here we have highlighted a novel mechanism(s) of endothelium-dependent and -independent vasocontraction to NADPH involving P2X receptors. As such, this study is the first to identify NADPH as a P2X receptor agonist. In addition, these findings should serve as a warning for future investigations that the effects of extracellular NADPH on vascular tone may not be solely attributable to NADPH oxidase activation.
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Footnotes |
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ABBREVIATIONS: ROS, reactive oxygen species; DETCA, diethyldithiocarbamic acid; SOD, superoxide dismutase; L-NAME, NG-nitro-L-arginine methyl ester; NF023, 8,8'-[carbonylbis (imino-3,1-phenylenecarbonylimino)]bis(1,3,5-naphthalene-trisulfonic acid); PPADS, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid; Ach, acetylcholine chloride; U46619
[GenBank]
, 9,11-dideoxy-11,9-epoxymethanoprostaglandin F2a; M40403, a manganese(II)-bis(cyclohexylpyridine)-substituted macrocyclic superoxide dismutase mimetic; Umax, maximum contraction to 0.3 µM U46619
[GenBank]
.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Grant Drummond, Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia. E-mail: grant.drummond{at}med.monash.edu.au
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, P < 0.05 versus endothelium-denuded; N.S., not significant versus control.
