Angiotensin-Converting Enzyme Inhibition Alters Nitric Oxide and Superoxide Release in Normotensive and Hypertensive Rats
Abstract Young (≈1 month old) male normotensive Wistar-Kyoto rats (n=26) and spontaneously hypertensive rats (n=38) were randomized into three groups treated via drinking water for ≈2 years with, respectively, placebo, low doses, or high doses of an angiotensin-converting enzyme inhibitor, ramipril (10 μg · kg−1 · d−1, non–blood pressure–lowering dose, or 1 mg · kg−1 · d−1, blood pressure–lowering dose). Relative to placebo treatment in each respective rat strain, both ramipril dosages increased endothelial constitutive nitric oxide synthase expression (Western blot) and resultant synthesis of nitric oxide (porphyrinic sensor) in freshly excised carotids and thoracic aortas, respectively. Paradoxically, this activity was associated with an increased/decreased superoxide accumulation (chemiluminescence) in freshly excised aortas from 24-/22-month-old normotensive/hypertensive rats. In normotensive rats, relative to placebo treatment, the threefold increase in superoxide accumulation with antihypertensive ramipril treatment is most likely from the >300% increase in endothelial constitutive nitric oxide synthase expression (some of which may be disarranged by local insufficiencies in l-arginine or tetrahydrobiopterin). In hypertensive rats, relative to placebo treatment, the 35% increase in nitric oxide availability by long-term antihypertensive ramipril treatment may contribute to the preservation of the endothelium and prevent its dysfunction by inhibiting superoxide production. Increased nitric oxide production with concomitant decreased superoxide accumulation (approximately one third of placebo levels) correlates positively with the previously reported +40% life span extension for rats with genetic hypertension that were treated with antihypertensive doses of ramipril.
The endothelium contributes in several ways to the local regulation of vascular tone by producing relaxing and contracting factors.1 Aging, per se, and hypertension are important risk factors of stroke and cardiovascular diseases and seem to be associated with endothelial dysfunction. However, endothelial dysfunction involves different mechanisms, depending on the vascular bed and the model of hypertension. It was suggested that in the aortas of SHR, the attenuated vasodilation in response to acetylcholine is most likely due to increased production of endothelium-derived cyclooxygenase-dependent contracting factors,2 while in the mesenteric artery of SHR, a similar reduced response was predominantly attributed to reduced production of NO.3 Recently it was shown, by direct measurement of NO in the mesenteric arteries of stroke-prone SHR, that calcium ionophore (A23187)–stimulated NO release is not impaired but is associated with accelerated decomposition of NO by O2−.4
In animals with genetic hypertension, subchronic inhibition of ACE was found to improve endothelium-mediated dilation of the thoracic aorta5 6 and the mesenteric artery.7 Moreover, early-onset, long-term treatment with ACE inhibitors was shown to prevent the impairment of endothelium-dependent vasodilation in SHR. This effect was associated with increased aortic cyclic GMP content.8 Preservation of endothelial function by ACE inhibitor treatment in rabbits on a long-term atherogenic diet was accompanied by enhanced aortic cyclic GMP content.9 In cultured endothelial cells, acute ACE inhibition led to enhanced production of intracellular cyclic GMP, which was strongly correlated to enhanced NO production.10 In clinical studies, it was documented that chronic ACE inhibition reduced the incidence of cardiovascular complications, especially myocardial reinfarction.11 12 13
This study describes comparative in vitro studies of the kinetics (patterns) of A23187-induced NO release from the aortas of normotensive WKY and SHR after early-onset (≈1 month after birth), long-term (≈2 years) treatment with ramipril, a known ACE inhibitor. The data obtained in these experiments were correlated with quantitative assessments of aortic O2− production. Ramipril increased NO release in both rat strains while diametrically altering the production of O2− in WKY and SHR. The increased NO production with concomitant decreased O2− production positively correlates with the +40% life span extension of ramipril-treated rats with genetic hypertension.14
Young (≈1 month old) male normotensive WKY (n=26) and SHR (n=38) with body weights of 75±3 and 72±3 g, respectively, and with systolic BP of 100±4 and 102±3 mm Hg, respectively, were obtained from Möllegaard, Skensved, Denmark. The animals were housed individually under control conditions (temperature, humidity, and light periods) and had free access to a standard diet (Altromint, maintenance diet 1320, sodium content 0.2%) and water ad libitum.
ACE Inhibitor Treatment
Both rat strains were randomized into one of the following groups: (1) without ramipril (placebo-treated; WKY, n=14; SHR, n=18); (2) ramipril-treated, 10 μg · kg−1 · d−1 (non–BP-lowering dose; WKY, n=6; SHR, n=10); and (3) ramipril-treated, 1 mg · kg−1 · d−1 (BP-lowering dose; WKY, n=6; SHR, n=10). Both rat strains were treated via drinking water for 23/21 months (WKY/SHR), respectively. Systolic BP was measured in conscious rats by tail plethysmography under light anesthesia.
Preparation of Thoracic Aortas
At 1, 8, and 24/22 months (WKY/SHR), selected rats were anesthetized with hexobarbital (80 mg/kg IP). The aorta was excised from each selected animal and carefully separated from adhering fat and connective tissue. Then, small segments from each thoracic aorta (5 mm behind the aortic arch) were carefully cut into rings (1 mm) and used in the experiments.
Expression of Endothelial cNOS
Densitometric analysis of Western blots was used to determine the relative expression of the endothelial cNOS in the carotid of WKY and SHR treated with ramipril for 23 and 21 months, respectively. Carotid arteries were excised freshly and washed immediately in PBS containing a protease-inhibitor cocktail (Complete, Boehringer Mannheim). The tissues were extracted on ice buffer containing 1% SDS, 10 mmol/L Tris-HCl, pH 7.4, and the protease-inhibitor cocktail. The supernatants were taken for further analysis. Total protein was determined from a small aliquot by using the Bio-Rad microassay according to the manufacturer’s instructions. Samples containing the same amounts of total protein were separated on SDS-polyacrylamide gel electrophoresis (10%) and transferred to Hybond-ECL nitrocellulose membranes (Amersham). The immunoblotting procedure was carried out using a 1:250 dilution of a specific monoclonal antibody to human endothelial cNOS (Transduction Laboratories) and the EL-Western blotting kit (Americium). Values are mean±SEM (n=6 animals per group).
Measurement of NO Release
NO was measured by using a porphyrinic microsensor that was interference free (at physiological conditions) from all reagents used in these experiments and all known readily oxidizable secretory products that may be found in mammalian tissue to at least two orders of magnitude greater than their expected concentrations.15 16 The porphyrinic microsensor had a response time of 0.1 millisecond at micromolar NO concentrations and 10 milliseconds at the detection limit of 1 nmol/L.
Preparation of the Porphyrinic Microsensor
The NO microsensor was produced by threading a single carbon fiber (Amoco Performance Products, 6 μm ID, 12 Ωcm) through the pulled end of an L-shaped (1 mm ID) glass capillary, until a 3-mm length of fiber protruded from the pulled capillary tip. A bare copper wire (AWG18, Arcor) covered with conductive silver epoxy (AI Technology) was inserted into the opposite end of the glass capillary and advanced as close as possible to the tip (2 to 3 mm before the tip) to insure good electrical contact with the active tip. Next, the tip of the glass capillary was sealed with beeswax. Excess beeswax was removed by immersion in 0.1 mol/L NaOH for 1 hour and rinsing with tap water and then distilled water.
The protruding carbon fiber tip of the porphyrinic sensor was made more sensitive to NO and less sensitive to potential interference by the cyclic voltammetric deposition (−0.20 to 1.00 V at 100 mV/s for 10 cycles) of a highly conductive polymeric porphyrin from a solution of 0.25 mmol/L nickel (II) tetrakis(3-methoxy-4-hydroxyphenyl) porphyrin16 in 0.1 mol/L NaOH, under nitrogen. Dip coating the dried catalyzed carbon fiber tip (3 times for 5 seconds) in 1% Nafion perfluorinated ion-exchange resin in alcohol (Aldrich) after drying produced a thin anionic film that repelled or retarded charged species while allowing small neutral and hydrophobic NO access to the underlying catalytic surface. Linear calibration curves were constructed for each sensor from 2×10−9 to 2×10−5 mol/L NO, before and after in vivo or in vitro measurements, using aliquots of saturated NO prepared as described.17
A porphyrinic microsensor working electrode, a platinum counterelectrode (0.5-mm diameter), and an SCE formed the three-electrode system used for NO measurement. Two methods of NO measurement, DPV and amperometry, were performed, using a Princeton Applied Research PAR model 273 voltammetric analyzer interfaced with an IBM 80486 computer with data acquisition and control software.
DPV was used to measure the basal NO concentration. Briefly, in the DPV method, current versus potential curves were generated in the potential range between 0.45 and 0.75 V versus SCE. The DPV peak current at the peak potential characteristic for NO oxidation (0.65 V) was found to be directly proportional to the local NO concentration in the immediate vicinity of the sensor.
Amperometry was performed at a constant applied potential (the peak potential for the oxidation of NO versus SCE). Amperometry was used for fast (resolution time 0.1 to 1 millisecond) and continuous measurement of the changes of NO concentration from its basal level with time.
The sensor was lowered with the help of a computer-controlled micromanipulator (resolution ±1 μm) until it reached the endothelial surface of an aortic strip (a small piezoelectric signal, 6 to 8 pA, of 1 to 3 milliseconds’ duration was observed at this point). The sensor was then slowly raised 4±1 μm from the surface of the aortic strip. The concentration of NO decreased exponentially with distance and was undetectable at distances higher than 130±20 μm from the endothelium. The sensor sampled only a small volume of solution (10−10 to 10−12 L) surrounding the sensor tip. Therefore, the concentration of NO measured was a local (close to the endothelial surface) concentration, not a bulk or global concentration.
Experimental Protocol for Measuring NO
Immediately before NO measurement, a longitudinally opened aortic ring was placed in an organ chamber with fresh phenol red–free HBSS, 5 mL, 37°C, pH 7.40 (Sigma), the active tip of the microsensor was placed on the endothelium of the vascular ring, and a platinum counterelectrode and reference SCE were placed on adjacent tissue. Then, 10 μL of 1.2 mmol/L solution of a calcium ionophore (A23187, Sigma) was injected to reach a final concentration of 8 μmol/L in the organ chamber, invoking maximal stimulation of cNOS. The experiment was then repeated in the presence of SOD (100 U/mL final concentration in the organ chamber, Sigma).
Indirect and Direct O2− Measurement
To determine indirectly the O2− concentration in aortic tissue sample, NO was stimulated with A23187 (8 μmol/L) as above, and its concentration was measured in the presence (Fig 1⇓, dotted line) and absence (Fig 1⇓, solid line) of SOD (100 U/mL). The difference in concentrations was assumed to be equal to the O2− concentration consumed in a reaction with NO.
To confirm the indirect O2− concentration data (Figs 1 through 3⇑⇓⇓), the O2− concentration in aortic tissue was directly determined by a chemiluminescence method (Fig 4⇓).18 O2−-produced chemiluminescence of lucigenin (bis-N-methylacridinium nitrate, Aldrich) was detected with a scintillation counter (Beckman 6000 LS, with a single photon monitor). Each (0.8- to 1.5-mg) tissue sample was placed in 2 mL of HBSS adjusted to pH 7.4, and lucigenin was added to reach a final concentration of 0.25 mmol/L. Basal O2− concentration produced by the tissue was measured after a 2-minute incubation in HBSS; photons were counted for 20 seconds. Total O2− production was measured after the 2-minute incubation in HBSS followed by injection of 10 μL of A23187 (1.2 mmol/L); photons were counted for 20 seconds after the addition of A23187. Calibration of O2− concentration was performed by constructing standard curves based on photons emitted by O2− stoichiometrically generated by reaction of xanthine and xanthine oxidase. Concentrations of O2− were reported in nanomoles per liter per milligram wet weight of the tissue.
To directly estimate the O2− production due to cNOS, basal and total O2− concentrations in aortic rings from desired treatment groups were incubated for 10 minutes with 20 μmol/L known inhibitors of cNOS, L-NAME and L-NMMA, both purchased from Sigma.19
The mean and SE of results were presented. Statistical evaluation was done by ANOVA followed by Student-Newman-Keuls test. Means were considered significantly different when the probability values were less than .05 (P<.05).
BP and Body Weight
Systolic BP was significantly lower in 8- and 24-month-old placebo-treated WKY than in the respective normotensive 8- and 22-month-old SHR. Treatment with 10 μg · kg−1 · d−1 or 1 mg · kg−1 · d−1 ramipril did not significantly change systolic BP in WKY. In SHR, 1 mg · kg−1 · d−1 ramipril reduced BP, whereas 10 μg · kg−1 · d−1 ramipril had no significant influence. Body weight was significantly more in 8- and 24-month-old untreated or placebo-treated WKY than in respective 8- and 22-month-old SHR. Ramipril treatment did not significantly affect the body weight of either group (Table⇓).
NO Release From Aortas of Untreated Animals
NO concentration released from the aortas of placebo-treated WKY and SHR, after stimulation with receptor-independent A23187 (8 μmol/L), was measured in vitro using a single-fiber porphyrinic sensor placed near the endothelium surface (4±1 μm). Amperometric curves showing the change of NO concentration with time, recorded in the absence and presence of SOD (100 U/mL), for young (1-month-old), adult (8-month-old), and senescent (24/22-month-old) placebo-treated WKY/SHR are depicted in Fig 1⇑. (Since SOD is a rapid scavenger of O2−, we used this indirect approach to estimate production of O2− at the time of NO release.)
After addition of A23187, a rapid increase of NO concentration was observed. Peak NO concentration was higher for young placebo-treated WKY than SHR (190±6 and 140±8 nmol/L, respectively; n=6, P<.005). Adult placebo-treated WKY and SHR showed similar patterns of NO release, peaking at 199±11 and 205±7 nmol/L, respectively. For senescent placebo-treated WKY and SHR, the peak concentration of NO was lower than in adult rats; the decrease was less for WKY (−4%) than for SHR (−17%).
In the presence of SOD, an increase of peak NO concentration was observed for all placebo-treated rats studied (Fig 1⇑). In the placebo-treated WKY strain, SOD treatment increased the peak NO release by 6% for both young and adult rats (17% and 9%, respectively, for SHR). In senescent placebo-treated WKY, the peak NO release was significantly higher, by 25.1%, after SOD treatment (from 191±6 to 239±10 nmol/L). In senescent placebo-treated SHR, the peak NO release was 53% higher after SOD treatment (from 175±6 to 269±10 nmol/L). SOD effectively scavenged only the protonated (HO2, pKa 4.8) portion of O2−, which can diffuse through the endothelial cell membrane. As expected, membrane-permeable O2− scavenger 4-hydroxy-TEMPO increased NO production 35% to 40% more than SOD. This finding indicates that a significant portion of O2− is protonated and can diffuse through the membrane and be scavenged by SOD.
NO Release From Aortas of Ramipril-Treated Animals
Long-term ACE inhibition with ramipril treatment increased the peak release of NO from freshly excised aortas of either strain of rat in a dose-dependent fashion: in 24-month-old WKY, from 191±6 nmol/L (placebo) to 258±22 nmol/L (low dose; non-antihypertensive) to 274±13 nmol/L (high dose; antihypertensive) (Fig 2⇑); in 22-month-old SHR, 175±6 nmol/L (placebo), 237±6 nmol/L (low dose), 404±22 nmol/L (high dose) (Fig 3⇑). Relative to placebo treatment, low-dose ramipril treatment increased the peak NO release 35% in both rat strains (Figs 2⇑ and 3⇑). Relative to placebo treatment, high-dose ramipril treatment affected the peak NO concentration differently: in WKY rats, there was a 43% increase (Fig 2⇑); in SHR, a 130% increase (Fig 3⇑).
In the presence of SOD, an increase of peak NO concentration was observed for all ramipril-treated rats studied (Figs 2⇑ and 3⇑). In ramipril-treated WKY, addition of SOD evoked a larger increase in NO release from freshly excised aortas than in similarly treated SHR aortas. The percentage increase of peak NO release in SHR by SOD treatment decreased from 32% to 8% as the dose of ramipril increased from 10 μg · kg−1 · d−1 to 1 mg · kg−1 · d−1, respectively.
The indirect measurement of O2− in aortic tissue obtained from the difference in NO measurement in the presence and absence of SOD or TEMPO analogue suggests that calcium ionophore A23187 not only stimulates NO release but also causes the simultaneous release of O2− (Figs 1 through 3⇑⇑⇑) and that this calcium-dependent O2− production varies with age (Fig 1⇑). This possibility was substantiated by use of a chemiluminescence method to make direct in vitro measurement of O2− in aortic tissue. Fig 4⇑ shows maximum concentration of O2−, directly measured in aorta of placebo-treated senescent WKY and SHR: basal concentration of O2− (Fig 4a⇑, directly measured in the absence of A23187) and total concentration of O2− (Fig 4b⇑, directly measured in the presence of A23187).
The total direct O2− concentration increased about three times, from 53±5 nmol · L−1 · mg−1 in placebo to 141±15 nmol · L−1 · mg−1 in ramipril (1 mg · kg−1 · d−1)-treated WKY (Fig 4b⇑). The total direct O2− production was reduced from 117±10 nmol · L−1 · mg−1 in placebo to 43±4 nmol · L−1 · mg−1 in high-dose (antihypertensive) ramipril-treated (1 mg · kg−1 · d−1) SHR (Fig 4b⇑). It is interesting to note that prolonged treatment with ramipril not only significantly increased NO concentration (Figs 2⇑ and 3⇑) but also diametrically changed basal and total concentration of O2− in WKY and SHR (Fig 4⇑).
As a negative control, 20-μmol/L doses of known inhibitors of cNOS, after a 10-minute incubation, were found to reduce directly measured calcium-dependent O2− release from both WKY and SHR (treated with placebo or high-dose ramipril) after A23187 stimulation, as above (Fig 4⇑). L-NAME inhibited production of O2− by about 25% (Fig 4⇑, solid bars). L-NMMA inhibited production of O2− stimulated by A23187 by about 70%. However, neither of these inhibitors reduced basal O2− release. L-NAME and L-NMMA reduced NO release (stimulated by A23187) by 35% and 40%, respectively.
Expression of cNOS Enzyme
Densitometric analysis of Western blots showed significant increase of the expression of cNOS in ramipril-treated WKY and SHR (Fig 5⇓). The relative increase of cNOS expression (treated with ramipril/placebo, n=6 animals per group) was highest (530±28%) for WKY and lowest (97±20%) for SHR treated with 10 μg · kg−1 · d−1 ramipril. The relative increase of cNOS expression was similar for both WKY and SHR treated with 1 mg · kg−1 · d−1 ramipril (361±12% and 353±8%, respectively; n=6 animals per group).
The present study documents, for the first time, the results of direct in situ measurements of maximally stimulated NO release and O2− production from the aortas of senescent WKY and SHR after early-onset (1 month after birth), long-term (23 and 21 months, respectively) treatment with the ACE inhibitor ramipril. To better judge the effects of nearly 2 years of ACE inhibitor treatment, we first investigated the influence of aging on NO release and O2− production by using 1-, 8-, and 24-/22-month-old WKY/SHR. As evident from data obtained by direct and continuous measurement of NO, after maximal A23187 stimulation, rapid increases of NO concentration in placebo-treated WKY and SHR strains were observed. In WKY, the peak NO release varies little with age. However, in young (1-month-old) SHR, the peak NO release was relatively low and increased with age, reaching a maximum in adult SHR (8 months old). This trend of increasing peak NO production with age was later reversed, resulting in a small decrease in senescent (24-month-old) WKY and a substantial decrease in senescent (22-month-old) SHR.
Additionally, the kinetics (patterns) of NO release were significantly different in WKY than in SHR. This difference is due to a simultaneous production of O2− that was much lower in WKY than SHR. The porphyrinic sensor detects only the net concentration of NO (ie, NO that is not consumed in fast chemical reaction and can freely diffuse to a target cell). This net concentration depends on not only the activity of cNOS and the substrates oxygen and Arg but also the concentration of O2− accumulated. The near-diffusion limited reaction of O2− with NO to form peroxynitrite (OONO−; k=3.8×109 L · mol−1 · s−1)20 is even faster than the reaction of O2− with SOD to form peroxide and oxygen (k=2×109 L · mol−1 · s−1).21 This observation suggests that the endogenous 5- to 10-mmol/L SOD in tissue cannot effectively prevent the nonenzymatic reaction of NO with O2− as long as basal concentrations of NO are maintained.20
When protonated (pKa 6.8), the peroxynitrous acid (HOONO) formed usually rapidly rearranges (t1/2<1 second) to form the relatively stable products hydrogen cation and nitrate anion.22 However, as the HOONO concentration increases as maximal O2− accumulations react with freshly synthesized NO (for example, during the initial stages of reperfusion after a prolonged ischemia), local HOONO concentration may become sufficient to assure its efficient transport to reactive sites as far as several cell diameters away.22 In the vicinity of certain reactive centers, HOONO may undergo homolytic cleavage to a hydroxyl free radical (OH) and a nitrogen dioxide free radical (NO2) or heterolytic cleavage to a nitronium cation (NO2+) and hydroxide anion (OH−).22 Three of these cleavage products, (OH, NO2 radicals, and NO2+) are among the most reactive and damaging species in biological systems and may initiate a cascade of events leading to the increase of cytotoxicity and decrease of life span of rats with genetic hypertension.14
Confirmation that cNOS rather than other sources produced most (≈70%) of the O2− came from experiments showing O2− production after treatment with known cNOS agonists, as well as its inhibition after incubation with known cNOS inhibitors. The rapid accumulation of O2− concentration in the presence of calcium ionophore A23187 suggests that production of O2− is calcium dependent as is the production of NO by cNOS.
Calcium-dependent generation of O2− by cNOS activity suggests a partial disarrangement of cNOS activity in the dysfunctional endothelium of SHR. We hypothesize that endothelial dysfunction may include hindered transport of Arg or H4B to the active site of cNOS or their local depletion, resulting in a disarrangement of cNOS.23 Furthermore, we hypothesize that activated cNOS may produce NO when the cNOS domains are arranged by sufficient local Arg or H4B, and O2− when its two domains are disarranged in the presence of insufficient local substrate Arg or labile cofactor H4B.24 Better-characterized enzymes with two domains have been shown to have substrate-arranged and substrate-deficient–disarranged conformations.25 Although no detailed crystallographic studies have been made on the effect of substrate or labile cofactors on cNOS conformation, it has been demonstrated with the isolated, purified enzyme that disarranged neuronal cNOS activity depends on the local concentration of Arg and H4B.24 The concentration of H4B in young SHR is slightly lower than in WKY (written communication from F. Cosentino, MD, December 1996) (both groups untreated with ramipril). Incubation of aortas of SHR with 10−6 to 10−5 mol/L H4B did not make a significant difference in the production of O2−. However, the incubation with 10−4 mol/L H4B decreased production of O2− by 15% to 20%. Therefore, local deficiency of Arg, as well as deficient concentrations of H4B, are probably responsible for disarrangement of endothelial NOS and high release of O2− in SHR.
The production of NO and O2− by disarranged cNOS activity in WKY and SHR can be inhibited by certain Arg derivatives. But it was quite surprising to observe that L-NMMA is more potent than L-NAME at inhibiting the production of O2− from aortic tissue after A23187 stimulation. A previous report on isolated Arg-starved neuronal cNOS found L-NAME effective but L-NMMA ineffective at inhibiting O2− production.19
Besides disarranged calcium-dependent cNOS activity, other calcium-independent sources contribute to the basal O2− production. In both WKY and SHR, we observed that the sum of the O2− produced by all these other sources accounts for only about 30% of the total O2− measured after addition of calcium ionophore A23187. The O2− produced by some other sources, such as xanthine oxidase, under physiological conditions, is efficiently scavenged by SOD or basal NO. However, this seems not to be the case in senescent SHR, in which a strongly enhanced basal O2− production was observed in contrast to WKY (Figs 3⇑ and 4a⇑). Under these conditions, the net released NO available for free diffusion to target cells decreases as a result of accelerated scavenging by O2−.
These findings are in accordance with earlier observations. In bioassay experiments, the basal release of endothelium-derived relaxing factor (now known to be NO) is decreased in intact aortas from 30- to 38-week-old SHR versus age-matched WKY27 ; and similarly, basal cGMP content is decreased in intact aortas from 15- to 18-month-old SHR versus age-matched WKY.28 Furthermore, in aortic segments and cardiac endothelium of adult SHR, basal NO synthase activity, determined by conversion of radiolabeled Arg to l-citrulline, is twofold to threefold enhanced versus that in adult WKY,29 30 probably as a compensatory mechanism to enhanced O2− production in hypertension. It has been suggested that in the aorta of SHR, enhanced O2− production by endothelial xanthine oxidase activity plays a more critical role in hypertension than decreased SOD activity.31
In addition, enhanced NO concentration not only rapidly scavenges O2− but may actually inhibit other enzymatic sources of O2−, such as NADPH oxidase.32 The depressed NO production in SHR caused an increase in O2− release from these same (non-cNOS) sources. This effect was clearly observed in this study; ie, the basal O2− (measured in the absence of calcium agonist) increased from 18±2 nmol · L−1 · mg−1 in WKY (24 months old) to 40±5 nmol · L−1 · mg−1 in SHR (22 months old), which show depressed NO release.
In both WKY and SHR, early-onset long-term treatment with ramipril enhanced the release of NO versus the respective placebo-treated animals. The increase in NO release by both doses of ramipril is supported by previously reported data8 that demonstrated in intact aortas from hypertensive rats that cGMP content increased not only after high-dose but also after low-dose ACE inhibitor treatment. Furthermore, the enhanced cGMP content after high- and low-dose ACE inhibitor treatment can be suppressed by cotreatment with a bradykinin receptor antagonist. Therefore, it is conceivable that in SHR, and most likely in WKY, the observed increases in NO release are mediated by inhibition of the breakdown of endothelium-derived kinins through ACE inhibition.33
It is interesting to note the diametrically different effects of ramipril treatment on the production of basal and total O2− by WKY and SHR (see Fig 4a⇑ and 4b⇑). In WKY, a threefold increase of total O2− production from 53±5 to 141±15 nmol · L−1 · mg−1 was observed. In contrast, in SHR, the original high O2− concentration (117±10 nmol · L−1 · mg−1) in placebo-treated SHR (in comparison with placebo-treated WKY) was reduced to almost one third of its original level (43±4 nmol · L−1 · mg−1) by ramipril treatment. The level of total O2− concentration in SHR after ramipril treatment is comparable to the O2− concentration of placebo-treated normotensive WKY.
To determine the mechanisms responsible for the decrease in O2− concentration in SHR and increase of O2− production in WKY by long-term treatment with ramipril will require further studies. Treatment with ramipril significantly increases the expression of cNOS, which correlates positively with increasing production of NO measured for both strains. However, in WKY, a significant fraction of the cNOS is probably disarranged due to lack of sufficient concentration of Arg or H4B manifested by a very high production of O2−, mostly by calcium-dependent cNOS. These data suggest that ramipril treatment causes an increase of cNOS expression in both rat strains, but apparently unlike SHR, in WKY this increased cNOS expression is not followed by a sufficient increase of Arg and/or H4B concentration.
Long-term ACE inhibition seems ultimately to prevent the development of disarranged catalytic activity of cNOS in SHR and thereby preserve the normal arranged cNOS activity in these animals, leading to higher net release of NO and lower production of O2− and HOONO. This situation will have a double beneficial effect on the cardiovascular system. Higher net production of NO will improve the vasodilation and anticoagulant properties of the cardiovascular system, and lower O2− and HOONO production may significantly decrease oxidative stress and extend the efficiency of the endothelium. These effects are surely major contributors to the significant extension of life span (+40%) observed in rats with genetic hypertension that were treated with an antihypertensive dose of ramipril.14
In summary, our results show that senescent normotensive and hypertensive rats are not associated with impaired NO synthesis and release but are associated with increased decay of NO by reaction with enhanced O2− accumulation from various sources, mainly from cNOS. Early-onset, long-term ramipril (an ACE inhibitor) treatment increases cNOS expression and resultant synthesis of NO in both rat strains. This effect is associated, paradoxically, with an increased/decreased oxidative stress in normotensive/hypertensive rats. In normotensive WKY, the enhanced O2− accumulation is most likely from elevated cNOS expression (some of which may be disarranged by local insufficiencies in Arg or H4B). In hypertensive rats, the enhanced NO availability by long-term ACE inhibition may contribute to the preservation of the endothelium and prevention of its dysfunction by inhibiting or scavenging O2− production, mainly from non-cNOS sources.
Selected Abbreviations and Acronyms
|cNOS||=||constitutive NO synthase|
|DPV||=||differential pulse voltammetry|
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
|SCE||=||saturated calomel reference electrode|
|SHR||=||spontaneously hypertensive rat(s)|
This work was supported in part by a grant from the Public Health Service (HL 55397) and the Research Excellence Fund, Institute of Biotechnology, Oakland University, Rochester, MI. We thank Dr Stephen Patton for helpful discussion.
- Received January 3, 1997.
- Revision received March 5, 1997.
- Accepted May 1, 1997.
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