Grant R. Drummond

Increased NADPH-Oxidase Activity and Nox4 Expression During Chronic Hypertension Is Associated With Enhanced Cerebral Vasodilatation to NADPH In Vivo

Background and Purpose— We examined the importance of NADPH-oxidase in reactive oxygen species production in cerebral arteries and its effect on vascular tone in vivo. Furthermore, we investigated whether chronic hypertension affects function or expression of this enzyme in cerebral vessels. Methods— Superoxide generation was detected in isolated rat basilar arteries with the use of lucigenin-enhanced chemiluminescence. mRNA expression of NADPH-oxidase subunits was assessed by real-time polymerase chain reaction. Basilar artery diameter was measured with the use of a cranial window preparation in anesthetized rats. Results— NADPH-stimulated superoxide production was 2.3-fold higher in arteries from spontaneously hypertensive rats (SHR) versus normotensive Wistar-Kyoto rats (WKY) and could be blocked by the NADPH-oxidase inhibitor diphenyleneiodonium. Higher NADPH-oxidase activity was also reflected at the molecular level as mRNA expression of the NADPH-oxidase subunit Nox4 was 4.1-fold higher in basilar arteries from SHR versus WKY. In contrast, expression of Nox1, gp91phox, p22phox, and p47phox did not differ between strains. Application of NADPH to basilar arteries caused larger vasodilatation in SHR than WKY. Vasodilatation to NADPH could be attenuated by diphenyleneiodonium, as well as diethyldithiocarbamate (Cu2+/Zn2+–superoxide dismutase inhibitor), catalase (H2O2 scavenger), or tetraethylammonium (BKCa channel inhibitor). Conclusions— Activation of NADPH-oxidase in cerebral arteries generates superoxide, which is dismutated by Cu2+/Zn2+–superoxide dismutase to H2O2. H2O2 then elicits vasodilatation via activation of BKCa channels. Upregulation of Nox4 during chronic hypertension is associated with elevated cerebral artery NADPH-oxidase activity.

Novel isoforms of NADPH oxidase in vascular physiology and pathophysiology

1.u2002Vascular cells have evolved to use reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, as signalling molecules. Under physiological conditions, ROS are important regulators of cell cycle, protein kinase activity and gene expression. However, in vascular disease states, such as hypertension and hypercholesterolaemia, excessive production of ROS may overwhelm the anti‐oxidant defence mechanisms of cells, resulting in ‘oxidative stress’, damage to the artery wall and, ultimately, development of atherosclerotic plaques.

Increased NADPH Oxidase Activity, gp91phox Expression, and Endothelium-Dependent Vasorelaxation During Neointima Formation in Rabbits

Reactive oxygen species including superoxide and hydrogen peroxide are important mediators in atherogenesis. We investigated the enzymatic source of vascular superoxide and its role in endothelium-dependent vasorelaxation during neointima formation. Silastic collars positioned around carotid arteries of rabbits for 14 days induced neointimal thickening. Using lucigenin-enhanced chemiluminescence, superoxide production was detectable in collared artery sections, but not in controls, only after inactivation of endogenous Cu2+/Zn2+-superoxide dismutase (Cu2+/Zn2+-SOD) with diethyldithiocarbamate (DETCA). Dihydroethidium staining indicated that endothelium and adventitia were the major sites of superoxide generation. Superoxide production in DETCA-treated collared arteries was enhanced further by NADPH and was inhibited by diphenyleneiodonium, suggesting NADPH oxidase was the source of the radical in collared arteries. Moreover, real-time PCR demonstrated 11-fold higher expression of the gp91phox subunit of NADPH oxidase in collared arteries than in controls. In vascular reactivity studies, endothelium-dependent vasorelaxation to acetylcholine did not differ between collared and control sections. However, treatment with DETCA reduced relaxations to acetylcholine in collared rings, but not in controls. NADPH further reduced relaxations to acetylcholine in DETCA-treated collared sections, but not in controls. In DETCA/NADPH-treated collared rings, sensitivity to nitroprusside, in contrast to acetylcholine, exceeded that of controls. Moreover, further treatment of such rings with exogenous Cu2+/Zn2+-SOD restored acetylcholine relaxations without altering nitroprusside responses. Thus, early neointimal lesions induced by periarterial collars are associated with elevated gp91phox expression and increased NAPDH-oxidase-dependent superoxide production in endothelium and adventitia. However, endothelium-dependent vasorelaxation is largely preserved due to the actions of Cu2+/Zn2+-SOD and increased smooth muscle sensitivity to nitric oxide.

Suppression of oxidative stress in the endothelium and vascular wall.

There is growing evidence that oxidative stress, meaning an excessive production of reactive oxygen and nitrogen species, underlies many forms of cardiovascular disease. The major source of oxidative stress in the artery wall is an NADPH oxidase. This enzyme complex in vascular cells, including endothelium, differs from that in phagocytic leucocytes in both biochemical structure and functions. The crucial flavin-containing catalytic subunits Nox1 and Nox4 are not present in leucocytes, but are highly expressed in vascular cells and upregulated in vascular remodeling, such as that found in hypertension and atherosclerosis. This offers the opportunity to develop vascular specific NADPH oxidase inhibitors that do not compromise the essential physiological signaling and phagocytic function carried out by reactive oxygen and nitrogen molecules. Although many conventional antioxidants fail to significantly affect outcomes in cardiovascular disease, targeted inhibitors of NADPH oxidase that block the source of oxidative stress in the vasculature are more likely to prevent the deterioration of vascular function that leads to stroke and heart attack.

Endothelium‐dependent relaxations mediated by inducible B1 and constitutive B2 kinin receptors in the bovine isolated coronary artery

1 Rings of bovine left anterior descending coronary artery (LAD) were contracted with the thromboxane A2‐mimetic, U46619 (1–30 nM), to approximately 40% of their maximum contraction to 125 mM KCl Krebs solution (KPSSmax) for comparison of responses to the B1 and B2 kinin receptor agonists, des‐Arg9‐bradykinin (des‐Arg9‐BK) and bradykinin (BK), respectively. Relaxation responses were normalized as percentages of the initial U46619‐induced contraction level, while contractile responses were expressed as percentages of KPSSmax. 2 After 6 h of in vitro incubation in Krebs solution at 37°C, des‐Arg9‐BK (pEC50, 8.00 ±0.08; maximum response (Rmax), 93.9 ±1.9%) and BK (pEC50, 9.75 ±0.07; Rmax, 100.1 ±0.7%) caused endothelium‐dependent relaxations in precontracted rings of bovine LAD which were competitively and selectively antagonized by the B. receptor antagonist, des‐Arg9‐[Leu8]‐BK (pA2, 6.27 ±0.11) and the B2 receptor antagonist Hoe‐140 (pA2, 9.63 +0.14), respectively. 3 At 3 h of in vitro incubation, the sensitivity (pEC50, 7.45 ±0.10) and Rmax (84.6 ±3.3%) to des‐Arg9‐BK were significantly less than those obtained in the same tissues at 6 h (pEC50, 7.94 ± 0.06; Rmax, 91.4±2.5%), whereas endothelium‐dependent relaxations to BK and ACh were unaffected by incubation time. 4 Relaxation responses to des‐Arg9‐BK, but not BK, at both 3 h and 6 h were significantly attenuated by the protein synthesis inhibitors, cycloheximide (30 and 100 μm) and actinomycin D (2 μm). 5 At 6 h, the nitric oxide (NO) synthase inhibitor, NG‐nitro‐L‐arginine (L‐NOARG, 100 μm), caused a significant 2 fold decrease in pEC50 (9.58 ±0.03) but had no effect on Rmax for BK. For des‐Arg9‐BK, L‐NOARG (100 μm) caused a marked and significant decrease in both the pEC50 and Rmax and revealed contractions to low concentrations of des‐Arg9‐BK. In both cases, L‐NOARG inhibition was reversed in the presence of L‐arginine (10 mM). 6 At 6 h, removal of the endothelium abolished relaxation responses to des‐Arg9‐BK and BK, and for des‐Arg9‐BK, but not BK, unmasked concentration‐dependent contractions (pEC50, 7.57 ±0.09; Rmax, 83.4±9.1%). The sensitivity of contractions to des‐Arg9‐BK increased slightly from 3h (pEC50, 7.37 ±0.08) to 6 h (pEC50, 7.62 ±0.12) of in vitro incubation; however, there was a small but significant depression in the maximum response over this time (Rmax, 126.8 ±8.5% and 103.3 ±8.6% for 3 h and 6 h of incubation respectively). 7 In conclusion, the bovine LAD contains inducible B1 and constitutive B2 endothelial cell kinin receptors, both of which mediate endothelium‐dependent relaxation partly via the release of NO. B1 receptors were also present on the smooth muscle layer of the bovine LAD.

Evidence for mediation by endothelium‐derived hyperpolarizing factor of relaxation to bradykinin in the bovine isolated coronary artery independently of voltage‐operated Ca2+ channels

1 The role of endothelium‐derived hyperpolarizing factor and voltage‐operated Ca2+ channels in mediating endothelium‐dependent, NG‐nitro‐L‐arginine (L‐NOARG; 100 μm) ‐resistant relaxations to bradykinin (BK), was examined in isolated rings of endothelium‐intact bovine left anterior descending coronary artery. 2 Rings of artery were contracted isometrically to approximately 40% or their respective maximum contraction to 125 mM KCl Krebs solution (KPSSmax) with the thromboxane A2‐mimetic, U46619. Relaxations to BK and the endothelium‐independent NO donor, S‐nitroso‐N‐acetylpenicillamine (SNAP), were normalized as percentages of reversal of the initial contraction to U46619. All experiments were carried out in the presence of indomethacin (3 μm). 3 BK caused concentration‐dependent relaxations [sensitivity (pEC50) 9.88±0.05; maximum relaxation (Rmax), 103.3±0.5%] in U46619‐contracted rings of bovine coronary artery. L‐NOARG (100 μm) caused a significant (P<0.01) 3 fold reduction in the sensitivity to BK (pEC50, 9.27±0.11) without affecting the Rmax (101.8±2.3%). A similar, significant 3 fold reduction in sensitivity to BK with no change in Rmax was observed after treatment with oxyhaemoglobin (20 μm; pEC50, 9.18±0.13, P<0.001) or a combination of oxyhaemoglobin (20 μm) and L‐NOARG (100 μm; pEC50, 9.08±0.10, P<0.001). Oxyhaemoglobin (20 μm) either alone or in combination with L‐NOARG (100 μm) caused an approximate 600 fold decrease in the sensitivity to SNAP. 4 The L‐type voltage‐operated Ca2+ channel inhibitor, nifedipine (0.3 μm‐3 μm), reduced the maximum contraction (Fmax) to isotonic 68 mM KCl Krebs solution (103.5±2.0% KPSSmax) by 85–90% (P<0.001); yet, the highest concentration of nifedipine (3 μm) caused only a small but significant reduction in both the sensitivity and Fmax to U46619. By contrast, nifedipine (3 μm) had no effect on the relaxation response to BK. Furthermore, a combination of nifedipine (3 μm) and L‐NOARG (100 μm) had no further inhibitory effects on relaxations to BK (pEC50, 8.79±0.10; Rmax, 101.7±2.4%) than did L‐NOARG (100 μm) alone (pEC50, 9.05±0.12; Rmax, 99.62±1.19). Also, nifedipine (0.3 μm and 3 μm) had no effect on the maximum relaxation to the K+ channel opener, levcromakalim (0.3 μm). 5 In the presence of nifedipine (0.3 μm to control contractions induced by high KCl) and isotonic 68 mM KCl Krebs solution (to inhibit K+ channel activity), relaxations to BK (pEC50, 9.42±0.10; Rmax, 93.9±1.8%) were similar to those observed in normal Krebs solution (pEC50, 9.58±0.09; Rmax, 98.4±0.8%). However, in the presence of 68 mM KCl Krebs solution the inhibitory effect of L‐NOARG (100 μm) on relaxations to BK (pEC50, 8.53±0.20; Rmax, 31.0±11.3%) was markedly greater than that in normal KCl Krebs solution (pEC50, 9.12±0.08; Rmax, 91.5±2.0%). Similar treatment with 68 mM KCl Krebs had no effect on relaxations to the NO donor, SNAP, yet abolished the response to the K+ channel opener, levcromakalim (0.3 μm). 6 In summary, this study has shown that (1) NO synthesis in response to BK in bovine coronary artery endothelial cells in situ is likely to be abolished by L‐NOARG, (2) NO‐independent relaxations to BK are markedly attenuated by 68 mM KCl‐containing Krebs, which, in the absence of L‐NOARG, had no effect, (3) nifedipine blocked contractions to a maximum‐depolarizing stimulus (KCl) yet had no effect on NO‐independent relaxations to BK, and (4) maximum relaxations to levcromakalim were abolished by 68 mM KCl Krebs but were not affected by nifedipine. Therefore, we hypothesize that if smooth muscle hyperpolarization is involved in non‐NO‐, endothelium‐dependent relaxation in bovine coronary arteries contracted with U46619, then it can accomplish this via a mechanism which does not involve closure of voltage‐operated Ca2+ channels.

SPECIAL REPORT Endothelium‐dependent relaxation to the B1 kinin receptor agonist des‐Arg ‐bradykinin in human coronary arteries

Des‐Arg9‐bradykinin (des‐Arg9‐BK) caused endothelium‐dependent relaxations in human, isolated coronary arteries which upregulated with in vitro incubation time. Relaxations to des‐Arg9‐BK were inhibited by the B1 receptor antagonist, des‐Arg9‐[Leu8]‐BK (pKB, 6.14 ± 0.11) but were unaffected by the B2 receptor antagonist, Hoe‐140. Therefore, this is the first demonstration that human coronary arteries possess endothelial B1 receptors which mediate endothelium‐dependent relaxation and appear to be synthesized de novo during the incubation period.

Reactive Oxygen Species in the Cerebral Circulation Physiological Roles and Therapeutic Implications for Hypertension and Stroke

It is now clear that reactive oxygen species (ROS) can act as signalling molecules in the cerebral circulation under both physiological and pathological conditions. Some major products of superoxide (O2•−) metabolism, such as hydrogen peroxide (H2O2) and hydroxyl radical (OH•), appear to be particularly good cerebral vasodilators and may, surprisingly, represent important molecules for increasing local cerebral blood flow.A major determinant of overall ROS levels in the cerebral circulation is the rate of generation of the parent molecule, O2•−. Although the major enzymatic source of O2•− in cerebral arteries is yet to be conclusively established, the two most likely candidates are cyclo-oxygenase and nicotinamide adenine dinucleotide phosphate (reduced form) [NADPH] oxidase. The activity of endogenous superoxide dismutases (SODs) play a vital role in determining levels and effects of all individual ROS derived from metabolism of O2•−.The term ‘oxidative stress’ may be an over-simplification that hides the complexity and diversity of the ROS family in cerebrovascular health and disease. Although a generalised increase in ROS levels seems to occur during several vascular disease states, the consequences of this for cerebrovascular function are still unclear.Because enhanced breakdown of O2•− by SOD will increase the generation of the powerful cerebral vasodilator H2O2, this latter molecule could conceivably act as a compensatory vasodilator mechanism in the cerebral circulation under conditions of elevated O2•− production.Some recent clinical data support the concept of a protective role for cerebrovascular NADPH oxidase activity. Although it is quite speculative at present, if NADPH oxidase were to emerge as a major source of beneficial vasodilator ROS in the cerebral circulation, this may represent a significant dilemma for treatment of ischaemic cerebrovascular conditions, as excessive NADPH oxidase activity is associated with the progression of several systemic vascular disease states, including hypertension and atherosclerosis.Despite data suggesting that antioxidant vitamins can have beneficial effects on vascular function and that their plasma levels are inversely correlated with risk of cardiovascular disease and stroke, the results of several recent large-scale clinical trials of antioxidant supplementation have been disappointing.Future work must establish whether or not increased ROS generation is necessarily detrimental to cerebral vascular function, as has been generally assumed, or whether localised increases in ROS in the vicinity of the arterial wall could be beneficial in disease states for the maintenance of cerebral blood flow.

Apamin-sensitive, non-nitric oxide (NO) endothelium-dependent relaxations to bradykinin in the bovine isolated coronary artery: no role for cytochrome P450 and K+

Since cytochrome P450‐derived metabolites of arachidonic acid and K+ have been implicated in endothelium‐derived hyperpolarizing factor (EDHF)‐dependent responses, the aim of this study was to determine whether such factors contribute to non‐nitric oxide (NO), endothelium‐dependent relaxation to bradykinin (BK) in bovine isolated coronary artery. In rings of artery contracted with U46619 and treated with indomethacin (3u2003μM) and NG‐nitro‐L‐arginine (L‐NOARG; 100u2003μM), relaxation to BK (0.01u2003nM‐0.3u2003μM) was blocked by ∼60% after inhibition of K+ channels with either high extracellular K+ (high [K+]o; 15–67u2003mM) or apamin (0.3u2003μM). Ouabain (1u2003μM), an inhibitor of Na+/K+‐ATPase, decreased the sensitivity to BK without affecting the maximum response. In L‐NOARG‐treated rings, ouabain had no further effect on the relaxation to BK. An inhibitor of inward‐rectifying K+ channels, Ba2+ (30u2003μM), had no effect on relaxations to BK in the absence or presence of either L‐NOARG or ouabain. KCl (2.5–10u2003mM) elicited small relaxations (∼20%) that were abolished by nifedipine (0.3u2003μM) and ouabain. Both the high [K+]o/apamin‐sensitive relaxation to BK, and the relaxation to the KATP channel‐opener, levcromakalim (0.6u2003μM), were unaffected by the cytochrome P450 inhibitor, 7‐ethoxyresorufin (10u2003μM), or by co‐treatment with a phospholipase A2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3; 3u2003μM) and a diacylglycerol (DAG)‐lipase inhibitor, 1,6‐bis‐(cyclohexyloximinocarbonylamino)‐hexane (RHC 80267; 30u2003μM). The non‐NO/high [K+]o‐insensitive, ∼40% relaxation to BK was, however, abolished by these treatments. Therefore, neither cytochrome P450‐derived metabolites of arachidonic acid nor K+ appear to mediate the EDHF‐like relaxation to BK (i.e the non‐NO, high [K+]o/apamin‐sensitive component) in bovine coronary arteries. Cytochrome P450‐derived metabolites may be released at higher BK concentrations to act in parallel with NO and the high [K+]o/apamin‐sensitive mechanism.