Role of Superoxide in the Depressed Nitric Oxide Production by the Endothelium of Genetically Hypertensive Rats
Abstract We undertook these studies to determine whether a deficient nitric oxide production in genetically hypertensive rats could result from its being scavenged by an excess production of superoxide. In one study we used a porphyrinic microsensor to measure nitric oxide concentrations released by cultured endothelial cells from stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY). SHRSP cells released only about one third the concentration of nitric oxide as did WKY cells. Treatment of cells with superoxide dismutase increased nitric oxide release, demonstrating that normally nitric oxide is scavenged by endogenous superoxide. The increase in nitric oxide release in response to superoxide dismutase treatment was more than twice as great from SHRSP as from WKY cells, demonstrating the greater amount of superoxide in the hypertensive rats. A direct measure of superoxide with the use of lucigenin demonstrated the presence of 68.1±7.1 and 27.4±3.5 nmol/L of this anion in SHRSP and WKY endothelial cells, respectively. The presence of superoxide in the rat aorta was also estimated by quantification of its effect on carbachol relaxation. This relaxation was diminished when endogenous superoxide dismutase was blocked by diethyldithiocarbamic acid. This blockade reduced the relaxation by 51.2±5.2% in SHRSP aortas and by only 22.0±8.2% (P=.015) in WKY aortas. Data from these diverse systems are in agreement that superoxide production is excessive in SHRSP tissues. This excess superoxide, by scavenging endothelial nitric oxide, could contribute to the increased vascular smooth muscle contraction and hence to the elevated total peripheral resistance of these rats.
Endogenous NO plays an important role in the regulation of blood pressure. This regulatory role is evident in the hypertension that occurs when the endogenous production of NO is prevented by blockade of NO synthase, the enzyme that produces NO.1 2 NO, of endothelial origin, normally maintains vascular smooth muscle in a partially relaxed state. When endogenous NO is eliminated, additional contraction of vascular smooth muscle occurs, resulting in an increase in vascular resistance and elevation in blood pressure.
We have reported that cultured endothelial cells from SHRSP produce less NO than do these cells from the WKY, the normotensive reference strain.3 This observation supports the hypothesis that an NO deficiency could contribute to the elevated arterial pressure in SHRSP. The hypothesis has clinical relevance because several investigators have reported a deficit in endothelial NO release in the brachial4 5 6 and coronary7 circulations in human essential hypertension. It should be noted, however, that two reports8 9 failed to observe this deficit.
The current study examined the possibility that there is in the endothelial cells in hypertension an excess superoxide radical (O2−) that scavenges NO as it is produced. In 1985 Wei et al10 concluded that “superoxide and other radicals … interfere with acetylcholine-induced endothelium-dependent vasodilation, probably because they destroy the endothelium-derived relaxant factor.” Gryglewski et al11 observed that the stability of the endothelium-derived relaxing factor, later established to be NO,12 was markedly increased by treatment with SOD. This observation led these authors to conclude that O2− is released by the endothelial cell along with endothelium-derived relaxing factor, inhibiting the action of this physiological vasodilator. They pointed out that these findings “suggest a central role for activated oxygen species in the pathogenesis of vasospasm, thrombosis and atherosclerosis.” It is now known that the reaction of NO and O2− results in the formation of peroxynitrite (ONOO−).13 Liu et al14 demonstrated that ONOO− is also a vasorelaxant and concluded that “the mechanism of superoxide inactivation of nitric oxide is by converting it to a shorter-lived and less potent vasorelaxant species.”
Recently, Ohara et al15 concluded that increased endothelial O2− production in hypercholesterolemic vessels may inactivate endothelium-derived NO and contribute to an early atherosclerotic process. We designed the current study to determine the role played by endothelial O2− in the deficient NO release that we had observed in endothelial cells from SHRSP.3 First, we determined the effect of SOD on NO release from cultured endothelial cells from WKY and SHRSP. In a second approach, we evaluated the effects of inactivating endogenous SOD with DETCA. This evaluation was carried out in a comparative study of carbachol relaxation of vascular smooth muscle from SHRSP and WKY.
Details of our methods for studying cultured cells were published earlier.3 In brief, endothelial cells were cultured from aortas of 5-week-old WKY and SHRSP. Use of these rats was approved for these studies by the University of Michigan Committee on Use and Care of Animals. The rats were from inbred colonies maintained at the University of Michigan for the past 16 years. NO released from endothelial cells in response to stimulation with bradykinin was monitored with a porphyrinic microsensor.16 Endothelial cells were grown to near confluence (3 or 4 days) in 35-mm Petri dishes. Culture medium was replaced with PSS. With the temperature maintained at 37°C, the porphyrinic sensor was micromanipulated through the PSS to the surface of the cells. Bradykinin was added to the PSS to give a concentration of 10−6 mol/L. The response was recorded as the peak concentration of NO resulting from this stimulation. The peak was reached within 2 minutes.
The concentration of O2− was determined by the method described by Gyllenhammar.17 O2− produced chemiluminescence of lucigenin (bis-N-methylacridimium nitrate), which was detected with a scintillation counter (Beckman 6000 LS, with a single photon monitor). Endothelial cells grown to near confluence in a 75-cm2 flask were scraped into 3 mL PSS. Lucigenin was added to this PSS to give a concentration of 0.25×10−3 mol/L. Photons were counted for 6 seconds immediately after this addition. Photon counts were calibrated as O2− concentration by constructing standard curves based on photons emitted by O2− generated in response to treating xanthine with xanthine oxidase. In this reaction O2− is produced stoichiometrically from xanthine. The chemical specificity of this light-yielding reaction for the O2− anion has been documented.17
Aorta: Isometric Tension Recording
Aortic rings were obtained from pentobarbital-anesthetized WKY and SHRSP. These rats were descendants of the two strains maintained at Michigan and used in the current study for the endothelial cell cultures; however, the rats used for these aortic tension recordings were from colonies that had been inbred in Glasgow for the past 3 years. Rings (2 to 3 mm) from the thoracic aortas of 16-week-old rats were mounted under 1 g tension in a 10-mL organ bath containing PSS of the following composition (mmol/L): NaCl 130, KCl 4.7, NaHCO3 14.9, KH2PO4 1.18, MgSO4-7H2O 1.17, CaCl2-2H2O 1.6, glucose 5.5, and CaNa2 EDTA 0.03. The PSS was aerated with 5% CO2/95% O2 and maintained at 37°C. After a 1-hour equilibration period the irreversible inhibitor of endogenous SOD, DETCA (10−2 mol/L),13 was added to some baths and vehicle to others (control). The inhibitor or its vehicle was washed from the bath in 45 minutes. Full concentration-response curves to phenylephrine were then constructed. These curves were similar for aortic rings from the four groups, with the following maximal responses to phenylephrine (10−5 mol/L) in grams of force developed: WKY control, 0.83±0.08; WKY DETCA–treated, 0.84±0.05; SHRSP control, 0.72±0.08; and SHRSP DETCA–treated, 0.77±0.07. The rings were then stimulated to contract to their individual EC50 concentrations before carbachol was added. This EC50 for all rings was approximately 3×10−7 mol/L. At the plateau of contraction the rings were made to relax with incrementing concentrations of carbachol (10−8 to 10−5 mol/L). In other studies SOD (45 U/mL) was added to the PSS 5 minutes before the aortic ring was made to contract with phenylephrine. The carbachol relaxation procedure was again carried out from the plateau of a phenylephrine contraction in the presence of SOD.
Chemicals and Statistical Analysis
SOD (bovine liver), lucigenin, DETCA, and all components of the culture media and PSS were obtained from Sigma Chemical Co.
For comparisons between WKY and SHRSP of NO and O2− released and carbachol relaxation, the unpaired Student’s t test was used. A value of P<.05 was considered to represent a statistically significant difference.
Any spontaneous release of NO that may have occurred from unstimulated endothelial cells (SOD-treated or not) failed to release an NO concentration that reached the limit of sensitivity (1 nmol/L) of the porphyrinic electrode. The NO concentration released from these cells in response to bradykinin (10−6 mol/L) stimulation is depicted in Fig 1⇓. These stimulated values were obtained from the PSS in either Petri dishes of untreated cells or from different Petri dishes of cells that had been treated (5 minutes) with one of several SOD concentrations (5 to 100 U/mL). When the untreated cells were stimulated with bradykinin, those from SHRSP released only one third the NO concentration as did those from WKY. Pretreatment of the cells with SOD resulted in an increase in NO release from both cell types. However, the increase in NO released was much greater from cells of SHRSP than it was from those of WKY. A maximal effective SOD concentration was achieved at approximately 100 U/mL. At this concentration the SHRSP cells released only about 20% less NO than did WKY cells.
The results of 11 of these studies are summarized in Fig 2A⇓. Whereas pretreatment with 100 U/mL SOD increased the NO concentration released from SHRSP cells by 68.1±7.1 nmol/L, this treatment increased the NO concentration released from WKY cells by only 27.4±3.5 nmol/L (Fig 2A⇓, third pair of bars).
This suggestion of a difference in the O2− concentration in the endothelial cells from these two sources was confirmed by our use of lucigenin to make direct measurements of the concentrations of the O2− anion. SHRSP cells contained this anion in a concentration of 70.3±7.1 nmol/L, whereas WKY cells contained only 37.8±4.1 nmol/L (n=6, P<.01) (Fig 2A⇑, fourth pair of bars).
In our second approach to evaluating O2− production in arteries from SHRSP and WKY, we studied NO released by aortic rings in response to stimulation with carbachol. NO released by endothelial cells of these rings was quantified as the magnitude of vascular smooth muscle relaxation produced by carbachol. As depicted in Fig 3⇓, under control conditions relaxation was less in rings from SHRSP than in those from WKY. With the maximal carbachol concentration these relaxations were 78.3±6.6% and 94.4±2.4%, respectively (P<.05). Rings from these rats were also studied after treatment with DETCA. This inhibitor of endogenous SOD11 permits the accumulation of O2−, which scavenges the NO released in response to carbachol. O2− accumulation can therefore be measured as the magnitude of the reduction in carbachol-induced relaxation produced by DETCA. When SHRSP rings were treated with DETCA, the magnitude of the relaxation produced by the highest concentration of carbachol was reduced by 51.2±5.2% (Fig 2B⇑). Treatment of WKY rings with DETCA reduced carbachol relaxation by only 22.0±8.2% (P=.015). This estimate of the relative amounts of O2− present in SHRSP and WKY tissues corresponds well with the relative concentrations of this anion found in cultured endothelial cells in the two rat strains in the first part of this study (compare Fig 2A⇑ and 2B⇑).
Relaxations observed as the concentration-response curves to carbachol of aortic rings pretreated with SOD (45 U/mL) did not differ from control relaxation curves to carbachol in rings from either WKY (n=7) or SHRSP (n=6) (data not shown).
Results of these studies indicate that the concentrations of superoxide produced by cultured endothelial cells or by aortic rings from SHRSP were greater than the concentrations produced by these structures from WKY. Important evidence establishing the physiological role of NO has been based on its being destroyed by O2− and stabilized by SOD.18 19 The reaction of NO with O2− to form peroxynitrite is extremely rapid.20 In the current study, although untreated endothelial cells from SHRSP produced an NO concentration only about one third as great as did those from WKY, most of this difference was eliminated when O2− was removed by SOD treatment. Therefore, it can be concluded that the major reason for the deficit in NO concentration is that although its rate of production is nearly normal, it is scavenged as it is produced by the excess O2−.
In the second part of this study the functional importance of this excess O2− in SHRSP is evident. When SOD, the endogenous pathway for the disposal of O2−, is blocked, the accumulation of this anion impairs vascular smooth muscle relaxation. This impairment of relaxation is twice as great in aortas from SHRSP as it is in those from WKY (Fig 2B⇑), suggesting that O2− accumulation is greater in the hypertensive vessel. Another possible reason for this greater effect of blockade of endogenous SOD in SHRSP than in WKY could be that SOD activity is greater in the hypertensive vessel. Several measurements of SOD activity in hypertension have been reported. Whereas Sharma et al21 found SOD content to be elevated in aortas of rabbits with coarctation hypertension, Vega et al22 reported that SOD is reduced in aortas of rats with renal hypertension and Ito et al23 found that SOD is reduced in the myocardium of genetically hypertensive rats (SHR). In another relevant study Chen et al24 found that SOD activity was depressed in neutrophils and red blood cells from patients with pregnancy-induced hypertension. Thus, there is no uniform support for an increased SOD activity in hypertension that could explain the observed greater effect of blocking SOD activity in SHRSP.
However, DETCA, the blocker of endogenous SOD, clearly reduces carbachol relaxation more in rings from SHRSP than it does in those from WKY (Figs 2B⇑ and 3⇑). Although our current study with aortic rings does not exclude the possibility that some of this strain difference may be caused by a higher SOD activity in SHRSP, the interpretation that this greater reduction is caused by a greater O2− production in SHRSP is in accord with the results of our studies with cultured endothelial cells from these two rat strains. In these cells we observed that exogenous SOD caused a greater increase in NO release (NO not scavenged by O2−) in SHRSP than in WKY cells (Figs 1⇑ and 2A⇑, third pair of columns). This indirect evidence for a greater O2− concentration in endothelial cells from SHRSP compared with those from WKY was confirmed when O2− was quantified directly with lucigenin (Fig 2A⇑, fourth pair of columns).
Even in the absence of the SOD blocker, relaxation is significantly less in SHRSP than WKY (Fig 3⇑). This observation suggests that in the unblocked vessels sufficient O2− is produced to scavenge some of the NO, reducing the relaxation produced by the carbachol stimulation. Such an excess of O2− in the brachial and coronary vascular beds in essential hypertension could account for the depressed acetylcholine vasodilatation observed in this condition.4 5 6 7
The findings of our current study are in accord with those of Nakazono et al.25 These investigators studied the effects of a special form of SOD that they had synthesized. When injected intravenously this SOD underwent transcellular transport in the endothelial cells. Blood pressure of SHR but not that of WKY was decreased significantly by this treatment. They concluded that O2− in and around the vascular endothelial cells may play a critical role in the pathogenesis of hypertension. Our results by more direct measurements have confirmed this possibility.
Selected Abbreviations and Acronyms
|PSS||=||physiological salt solution|
|SHR||=||spontaneously hypertensive rat(s)|
|SHRSP||=||stroke-prone spontaneously hypertensive rat(s)|
Work by the authors is supported by British Heart Foundation grants Nr 92100 and 93025 to Anna F. Dominiczak, who is a British Heart Foundation Senior Research Fellow; by National Institutes of Health grants HL-46402 and HL-18575 (David F. Bohr); and by Biotechnology Research Program, Oakland University (Tadeusz Malinski). The authors are indebted to Leslie Turner for her skillful preparation of this manuscript.
- Received July 25, 1995.
- Revision received August 22, 1995.
- Accepted September 19, 1995.
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