Hui Di Wang

Superoxide Anion From the Adventitia of the Rat Thoracic Aorta Inactivates Nitric Oxide

The purpose of this study was to determine whether superoxide anion is produced endogenously in the rat aortic adventitia and whether sufficient superoxide anion is produced to interfere with the response of the rat aorta to nitric oxide. Relaxation was measured in rings of the rat thoracic aorta, which were oriented so that the adventitial or luminal surface could be preferentially exposed to nitric oxide or sodium nitroprusside. To accomplish this, the rings were mounted (1) with the adventitia facing outward, (2) with the adventitia facing inward after inverting, or (3) with the adventitia facing outward after inverting twice (to control for the inverting procedure). The relaxation to nitric oxide, but not to sodium nitroprusside, was less in rings with the adventitia facing outward compared with those in which it faced inward. In contrast, the response to nitric oxide via either surface was similar when extracellular superoxide anion was scavenged with superoxide dismutase. Incubation of rings with nitro blue tetrazolium (NBT) resulted in blue formazan staining of the adventitia, and lucigenin chemiluminescence was significantly greater when detected from the adventitial compared with the intimal aspect of the artery. The reduction of NBT in intact aortic rings was 30+/-2 pmol x min(-1) x mg(-1) and was significantly decreased by superoxide dismutase to 19+/-2 pmol x min(-1) x mg(-1) and by a synthetic superoxide dismutase mimic, Euk-8, to 11+/-2 pmol x min(-1) x mg(-1). The NADPH oxidase inhibitor, diphenyleneiodonium, decreased NBT reduction to 9+/-1 pmol x min(-1) x mg(-1), whereas inhibitors of xanthine oxidase, mitochondrial oxidases, and nitric oxide synthase were ineffective. Immunohistochemical staining indicated the localization of NADPH oxidase proteins gp91phox, p22phox, p47phox, and p67phox almost exclusively in the adventitia of the rat aorta with no substantial staining in the media. These results indicate that NADPH oxidase located in the adventitia of rat thoracic aorta generates sufficient extracellular superoxide anion to constitute a barrier capable of inactivating nitric oxide. This study suggests that adventitial superoxide anion can play a role in the pathophysiology of the arterial wall.

Paracrine Role of Adventitial Superoxide Anion in Mediating Spontaneous Tone of the Isolated Rat Aorta in Angiotensin II-Induced Hypertension

The relationship between vascular generation of superoxide anion and spontaneous tone observed in the isolated aorta was studied in hypertensive rats infused with angiotensin II. Aortic rings from hypertensive, but not from sham-operated rats, demonstrated oscillatory spontaneous tone that represented 52+/-5.6% of the maximal contraction to KCl. Spontaneous tone was prevented by calcium-free buffer or by blocking calcium influx through L-type calcium channels with nifedipine. The production of superoxide anion measured by lucigenin chemiluminescence was up to 15-fold higher than in sham-operated rat aorta. The adventitial site of production of superoxide anion was suggested by the fact that lucigenin chemiluminescence was 5.5-fold higher from the adventitia than from the intima. This was confirmed histochemically by demonstrating that the adventitia was the site of reduction of nitroblue tetrazolium as well as immunohistochemical staining of NAD(P)H oxidase subunit proteins. A causal link between superoxide anion production by NAD(P)H oxidase and the spontaneous tone is suggested by the fact that superoxide dismutase or the inhibitor of NAD(P)H oxidase, diphenylene iodonium, decreased both superoxide anion production and spontaneous tone. L-NAME or removal of the endothelium from the aorta had no significant effect on superoxide anion levels or spontaneous tone. However, although superoxide dismutase decreased superoxide anion levels in the presence of L-NAME or in endothelium-denuded rings, it no longer inhibited the tone. This suggests that the effect on tone of superoxide anion originating in the adventitia is mediated by inactivating endothelium-derived nitric oxide, which promotes smooth muscle calcium influx and spontaneous tone. The adventitia is not a passive bystander during the development of hypertension, but rather it may have an important role in the regulation of smooth muscle tone.

Effect of an Endothelin Antagonist on Hemodynamic Responses to Angiotensin II

We determined changes in blood pressure, cardiac output, and total peripheral conductance evoked by intravenous infusions of angiotensin II (Ang II) in conscious, unrestrained normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) before and after pretreatment with bosentan, a nonselective endothelin antagonist. Blood pressure was recorded by radiotelemetry and cardiac output by ultrasonic transit-time flow probes. Bosentan per se failed to affect basal blood pressure and evoked only small changes in cardiac output and total peripheral conductance in both strains. The pressor effects of Ang II were exaggerated in SHR compared with WKY. Strikingly, bosentan pretreatment blunted the increases in blood pressure, the fall in cardiac output, and the decreases in conductance evoked by lower doses of Ang II but not higher doses of the peptide. This effect was observed in both rat strains but was more pronounced in SHR. These data suggest that endothelin contributes to the hemodynamic effects of Ang II in both SHR and WKY and that endothelin may contribute to the exaggerated pressor responsiveness of SHR to Ang II.

Role of oxidative stress and nitric oxide in regulation of spontaneous tone in aorta of DOCA-salt hypertensive rats

The roles of nitric oxide (NO), superoxide anion (O2−), and hydrogen peroxide (H2O2) in the modulation of spontaneous tone were investigated in isolated aorta from deoxycorticosterone acetate (DOCA)‐salt hypertensive rats. Increases in preload from 1 to 5 g were accompanied by increases in spontaneous tone in aortic rings from DOCA‐salt hypertensive rats but not from SHAM‐normotensive rats. Tone was higher in endothelium‐denuded aortic rings than in endothelium‐intact vessels. Inhibition of nitric oxide synthase (NOS) with 300 μM NG‐nitro‐L‐arginine methyl ester (L‐NAME) increased spontaneous tone. Basal O2− generation was higher in aortic rings from DOCA‐salt hypertensive rats than in those from SHAM‐normotensive rats. Stretch increased O2− levels even further in the DOCA‐salt group. In rings isolated from DOCA‐salt hypertensive rats, administration of the O2− scavenger, superoxide dismutase (SOD, 150 U ml−1), or the nicotinamide adenine dinucleotide phosphate (NADPH)‐oxidase inhibitor, apocynin (100 μM), completely abolished the development of spontaneous tone in endothelium‐intact aortic rings but not in endothelium‐denuded or in L‐NAME‐treated rings. SOD and apocynin decreased the generation of O2− in endothelium‐intact, endothelium‐denuded, and L‐NAME‐treated aortic rings. Oral treatment of DOCA‐salt hypertensive rats with the O2− scavengers, tempol or tiron, or with apocynin for 3 weeks prevented the development of hypertension and abolished the increases in O2− generation and spontaneous tone. Administration of catalase (1000 U ml−1) to aortic rings increased spontaneous tone in vessels from DOCA‐salt hypertensive rats. Administration of the cyclooxygenase (COX) inhibitor, valeroyl salicylate, or the thromboxane/prostaglandin antagonist, SQ 29548, to aortic rings abolished tone. The results suggest that NO plays a major role in preventing the generation of spontaneous tone in isolated aortic rings from DOCA‐salt hypertensive rats. NADPH‐oxidase‐derived O2− enhanced spontaneous tone by inactivating NO. Endogenous H2O2 appears to mitigate the increase in tone. In addition, a COX component may also contribute to spontaneous tone.

Tempol selectively attenuates angiotensin Ii evoked vasoconstrictor responses in spontaneously hypertensive rats

Objective To assess whether superoxide anions mediate vasoconstrictor responses to agonists in blood vessels of spontaneously hypertensive rats (SHRs). Methods The effect of the superoxide dismutase mimetic, 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol), on responses to angiotensin II (Ang II), endothelin-1, phenylephrine and potassium chloride was determined in aortic rings and perfused mesenteric vascular beds (MVB) of adult male rats of the Sprague–Dawley, Wistar–Kyoto (WKY) and spontaneously hypertensive (SHR) strains. The effect of tempol on Ang II-evoked superoxide production was assessed in aortic rings. Results There were no differences in the maximum tension (Emax) attained in response to agonists, but the negative logarithm of the concentration required to produce 50% of the maximal response (EC50) for Ang II was lower (P < 0.05) in aortic rings of SHRs. In the MVBs of SHRs, the Emax but not the EC50 values attained in response to Ang II, endothelin-1 and phenylephrine were greater. Tempol significantly and selectively reduced the Emax of Ang II in both aorta and MVB preparations with intact endothelium. The reduction in Emax attained in response to Ang II was more pronounced in SHRs (P < 0.01) than in WKY rats (P < 0.05) or Sprague–Dawley rats (P < 0.05). The inhibitory effect of tempol was absent when a nitric oxide synthase inhibitor was included or endothelium was denuded. A significant increase in lucigenin chemiluminescence evoked by Ang II in both intact and endothelium-denuded aortic rings of SHRs was abolished when tempol was included in the buffer. Conclusions These data suggest that increased superoxide anions mediate vasoconstrictor responses to Ang II, but not to other agonists, in an endothelium-dependent manner, by quenching vasodilatory mediator, nitric oxide. This may account for the exaggerated vasoconstrictor responses to Ang II in SHRs.

ETA and ETB receptors are expressed in vascular adventitial fibroblasts

The adventitia has been recognized to play important roles in vascular oxidative stress, remodeling, and contraction. We recently demonstrated that adventitial fibroblasts are able to express endothelin (ET)-1 in response to ANG II. However, it is unclear whether ET-1 receptors are expressed in the adventitia. We therefore investigated the expression and roles of both ET(A) and ET(B) receptors in collagen synthesis and ET-1 clearance in adventitial fibroblasts. Adventitial fibroblasts were isolated and cultured from the mouse thoracic aorta by the explant method. Cultured cells were treated with ANG II (100 nmol/l) or ET-1 (10 pM) in the presence or absence of the ANG II type 1 receptor antagonist losartan (100 μM), the ET-1 receptor antagonists BQ-123 (ET(A) receptor, 1 μM) and BQ-788 (ET(B) receptor, 1 μM), and the ET(B) receptor agonist sarafotoxin 6C (100 nM). ET-1 peptide levels were determined by ELISA, whereas ET(A), ET(B), and collagen levels were determined by Western blot analysis. ANG II increased ET-1 peptide levels in a time-dependent manner. ANG II increased ET(A) and ET(B) receptor protein levels as well as collagen in a similar fashion. ANG II-induced collagen was reduced while in the presence of BQ-123, suggesting a role for the ET(A) receptor in the regulation of the extracellular matrix. ANG II treatment in the presence of BQ-788 significantly increased ET-1 peptide levels. Conversely, the ET(B) receptor agonist sarafotoxin 6C significantly decreased ET-1 peptide levels. These data implicate a role for the ET(B) receptor in the clearance of the ET-1 peptide. In conclusion, both ET(A) and ET(B) receptors are expressed in adventitial fibroblasts, which paves the ground for the biological significance of adventitial ET-1. The ET(A) receptor subtype mediates collagen I expression, whereas the ET(B) receptor subtype may play a protective role through increasing the clearance of the ET-1 peptide.