In the aorta of male spontaneously hypertensive rats (SHR), but not in that of normotensive Wistar-Kyoto rats (WKY), contractions to phenylephrine obtained in the presence of L-NAME [inhibitor of nitric oxide synthase (NOS)] and indomethacin (inhibitor of cyclooxygenase) are inhibited by an unknown endothelium-derived factor. The present study aimed to identify the mechanism underlying this endothelium-dependent inhibition in the SHR aorta. Aortic rings of male SHR and WKY, with and without endothelium, were suspended in organ chambers in the presence of indomethacin and L-NAME for the measurement of isometric tension. Contractions to phenylephrine were smaller in SHR aortae with endothelium than in those without, but were similar in the two types of preparations of WKY aortae. The endothelium-dependent, NOS-independent inhibition of phenylephrine-induced contraction was abolished by oxyhemoglobin [extracellular NO scavenger], carboxy-PTIO (NO scavenger) and ODQ (inhibitor of soluble guanylyl cyclase). It was unmasked not only by indomethacin but also by apocynin (antioxidant), but inhibited by diphenyleneiodonium (inhibitor of flavoproteins including cytochrome P450 reductase). The cytochrome P450 reductase protein expression was similar in SHR and WKY aortae. However, the level of nitrate and nitrite, substrates of cytochrome P450 reductase, were higher in SHR than WKY plasma and aortae. Therefore, in SHR but not WKY aortae, eNOS-independent NO is formed by cytochrome P450 reductase.
The endothelium modulates vascular tone by releasing endothelium-derived relaxing and contracting factors (Furchgott and Vanhoutte 1989; Vanhoutte et al., 2009). Among the relaxing factors, prostacyclin and NO play a prominent role (Moncada et al., 1976; Gruetter et al., 1979; Furchgott and Zawadzki, 1980; Furchgott, 1993). Prostacyclin is formed by endothelial cyclooxygenases, diffuses to the underlying vascular smooth muscle cells, and activates IP receptors, which in turn stimulate adenylyl cyclase to produce cyclic adenosine monophosphate, thereby inhibiting the contractile process (Moncada et al., 1978). Endothelium-derived NO diffuses to the vascular smooth muscle and stimulates soluble guanylyl cyclase to produce cyclic guanosine monophosphate, causing relaxation (Gruetter et al., 1979; Palmer et al., 1987; Palmer et al., 1988; Furchgott, 1993).
Endogenous NO is formed by both enzymatic and nonenzymatic reactions in the cardiovascular system. NO has a short half-life as it is scavenged by superoxide anions (Rubanyi and Vanhoutte, 1986) or quickly oxidized by O2, reactive oxygen species (ROS), and hemoglobin to form nitrate or nitrite (Chen et al., 2008). A major mechanism of NO formation is by nitric oxide synthase (NOS) [including neuronal (nNOS; NOS I), inducible (iNOS; NOS II) and endothelial (eNOS; NOS III) isoforms], which involves a series of redox reactions, with L-arginine being the substrate and L-citrulline and NO the end-products (Palmer et al., 1988; Alderton et al., 2001). Although eNOS accounts for the majority of NO produced in the vasculature, other sources have been identified. Thus, the nitrate and nitrite in the blood and extra vascular tissues can be reduced to form NO under anoxic or acidotic conditions by different enzymes located in various tissues (Li et al., 2006).
Preliminary studies yielded the unexpected finding that contractions of the aortae of spontaneously hypertensive rats (SHR) to phenylephrine were reduced in the presence of the endothelium (Fig. 1) despite the presence of the inhibitors of the major endothelium-dependent relaxation pathways, L-NAME and indomethacin (in concentrations supposed to block eNOS and cyclooxygenases, respectively). The present experiments were designed to determine the nature of this endothelial inhibitory signal.
Materials and Methods
The study was approved by the Committee on the Use of Live Animals in Teaching and Research of The University of Hong Kong. The experiments were conducted in male, 36- to 46-week-old SHR and normotensive Wistar Kyoto rats (WKY). The animals were purchased from the Laboratory Animal Services Centre of the Chinese University of Hong Kong and housed in the Laboratory Animal Unit of the University of Hong Kong in a temperature-controlled room (21°C ± 1°C) with a 12-hour light/dark cycle (0700 h lights on, 1900 h lights off). They had free access to chow and water. They were euthanized with an overdose of pentobarbital sodium (140 mg/kg) and exsanguinated.
Aortae were dissected free, excised, and placed in cold, modified Krebs-Ringer bicarbonate solution of the following composition (in mM): NaCl (118), KCl (4.7), CaCl2 (2.5), MgSO4 (1.2), KH2PO4 (1.2), NaHCO3 (25), and glucose (5.5) (control solution). They were cut into rings (3–4 mm in length). In some preparations, the endothelium was removed mechanically by inserting the tip of a syringe needle into the ring and rolling it back and forth in a container filled with control solution.
Organ Chamber Studies.
The rings were suspended in organ chambers, which contained control solution (37°C), aerated with 95% O2 and 5% CO2. They were connected to force transducers (FT03; Grass Instrument, Quincy, MA) for isometric tension recording (PowerLab ADInstruments, Sydney, NSW). The rings were allowed to equilibrate for 2 hours at optimal resting tension of 2.5 g (determined in preliminary experiments, data not shown). Changes in tension were expressed as percentages of the reference contraction to KCl (60 mM) obtained at the start of the experiment.
Measurement of Total Nitrate and Nitrite Levels.
The plasma was collected before sacrifice. Aortae were homogenized in ice-cold reaction buffer and centrifuged at 10,000g for 15 minutes at 4°C; the supernatant was used for the measurements. The total nitrate and nitrite concentration was determined using a total nitric oxide assay kit (Enzo Life Sciences, Farmingdale, New York, NY).
Fluorescence of DAF-FM-NO.
Human aortic endothelial cells (HAEC) were cultured on cover glass in wells using Medium 200 (Invitrogen, Carlsbad, CA). The medium was changed to Medium 200PRF (Invitrogen) 24 hours before the experiment. Cells were washed with control solution and incubated with different pharmacological inhibitors for 30 minutes. They were then loaded with DAF-FM diacetate (10−6 M) for 30 minutes at 37°C. After washing away the excess DAF-FM diacetate, cells were stimulated with A23187 (10−6 M), sodium nitrate (10−4 M) or sodium nitrite (10−4 M) for 20 minutes. Following fixation and mounting, fluorescence of the cells was detected with a fluorescence microscope BX41 (Olympus, Tokyo, Japan) at 495/515 nm.
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), acetylcholine chloride, allopurinol, daidzin, indomethacin, Nω-nitro-L-arginine methyl ester (L-NAME) and phenylephrine were purchased from Sigma-Aldrich (St. Louis, MO). Carboxy-PTIO, clotrimazole (CLT), diphenyleneiodonium (DPI) and PGE2 were purchased from Cayman Chemical (Ann Arbor, MI). Apocynin was purchased from Avocado Research Chemicals (Heysham, UK). N-[[3-(Aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride (1400W) was purchased from Enzo Life Sciences (Farmingdale, NY). Oxyhemoglobin was made from hemoglobin (Sigma) and prepared as described (Martin et al., 1985). Stock solutions of CLT and DPI were prepared in absolute dimethyl sulfoxide (DMSO). A stock solution of indomethacin was prepared in a sodium bicarbonate solution (5 ×10−3 M). ODQ, apocynin, and prostaglandin E2 were prepared in ethanol. DAF-FM diacetate (Invitrogen, Carlsbad, CA) was prepared in DMSO. All other compounds were prepared in deionized water. Concentrations are expressed as final molar concentrations in the organ chambers or in the cell culture wells. The highest concentration used of DMSO was 0.02% in the organ chambers (control to CLT and DPI) and in cell culture wells; the highest concentration of ethanol was 0.3% in organ chambers (control to apocynin). They had no significant vascular effect. The concentrations used of the inhibitors were selected from earlier work in the laboratory or from the literature.
Results are presented as means ± S.E.M.; n refers to the number of experimental animals. The effects of CLT and DPI on the responses to phenylephrine were analyzed by means of the area under the concentration-response curve, which was calculated using the statistical software Prism version 5 (GraphPad Software, La Jolla, CA). Statistical analysis was done by Student’s t test and two-way ANOVA, using Prism version 5. P values less than 0.05 were considered to indicate statistically significant differences.
In the presence of L-NAME (10−4 M) and indomethacin (10−5 M), contractions to phenylephrine were significantly smaller in SHR aortae with endothelium than in those without but were similar in the two types of preparations of WKY aortae (Fig. 1A). Contractions evoked by PGE2 were also smaller in SHR aortae with endothelium than in those without (Fig. 1B). The difference in phenylephrine-induced contractions between SHR aortic rings with and without endothelium were abolished by carboxy-PTIO [3 × 10−4 M (Rand and Li, 1995)] and oxyhemoglobin [10−6 M (Feelisch and Noack, 1987)], cell permeable and cell impermeable NO scavengers, respectively (Fig. 2, A and B), and by ODQ [10−5 M; soluble guanylyl cylase inhibitor; Fig. 2C]. Contractions of WKY aortic rings with and without endothelium were not affected by carboxy-PTIO, hemoglobin or ODQ (Fig. 2D).
L-NAME (10−4 M) abolished the relaxations to acetylcholine (Fig. 3A) in SHR aortae with endothelium. The selective inhibitor of iNOS 1400W [10−5 M (Topal et al., 2012)] did not affect the difference between rings with and without endothelium (Fig. 3B).
Incubation with indomethacin impaired the response to the α1-adrenoceptor agonist, an effect that was prevented not by L-NAME but by the nitric oxide scavenger oxyhemoglobin (Fig. 4A). Meclofenamate (10−6 M; data not shown) and apocynin [3 × 10−4 M; antioxidant (Heumüller et al., 2008)] mimicked the effect of indomethacin (Fig. 4B).
Sodium nitrate relaxed SHR aortae with, but not those without endothelium. Sodium nitrite caused similar relaxations in SHR aortae with and without endothelium (Fig. 5).
Diphenyleneiodonium and Clotrimazole.
Diphenyleneiodonium [DPI; 3 × 10−7 M; inhibitor of flavoproteins, including cytochrome P450 reductase (McGuire et al., 1998)] reduced contractions to phenylephrine in SHR rings without endothelium but significantly augmented them in the presence of endothelium (Fig. 6, A and D). In the presence of DPI, phenylephrine-induced contractions were not significantly different in rings with and without endothelium (Fig. 6, A and D). Clotrimazole (CLT; 10−6 M; cytochrome P450 inhibitor) did not affect phenylephrine-induced contractions in rings either with or without endothelium (Fig. 6, B and E), and it did not modify the effect of DPI on the response to the α1-adrenoceptor agonist (Fig. 6, C and F).
Unlike DPI, allopurinol (5 × 10−5 M, xanthine oxidoreductase inhibitor, Fig. 7A) and daidzin (10−5 M, aldehyde dehydrogenase 2 inhibitor, Fig. 7B) had no effect on the difference in phenylephrine-induced contractions between SHR aortic rings with and without endothelium in the presence of indomethacin and L-NAME.
Expression of Cytochrome P450 Reductase and Total Nitrate and Nitrite.
The protein expression level of cytochrome P450 reductase was similar in SHR and WKY aortae (Fig. 8). The concentrations of total nitrate/nitrite in plasma and aortae of SHR were significantly higher than in those of WKY (Fig. 9).
Nitric Oxide Production in HAEC.
The fluorescence due to binding of NO to DAF-FM diacetate increased in HAEC exposed to A23187, nitrate, and nitrite (Fig. 10). The nitrate- and nitrite-induced increases in fluorescence were reduced in cells treated with CLT and DPI.
Aortic rings of SHR and WKY without endothelium exhibited similar contractions to phenylephrine; however, in the presence of L-NAME, the endothelium impaired contractions to the α1-adreneceptor agonist only in preparations of the hypertensive strain. Aortic rings without endothelium of SHR exhibited higher contractions to prostaglandin E2 than those of WKY. Contractions to prostaglandin E2 in the rat aorta are due to activation of both EP1 and TP receptors (Tang et al., 2008). The vascular smooth muscle of the SHR aorta is hyperresponsive to TP agonists (Ge et al., 1995), as confirmed in the present study. Despite the presence of NOS inhibitors, the endothelium of the SHR aorta attenuated the contraction to the prostanoid to the same level as in that observed in WKY preparations. Thus, the present study demonstrates that, under conditions where the production of NO by NOS is excluded, contractions in SHR aortae are inhibited by the presence of the endothelium in a nonselective manner. The endothelium-derived factor involved is present only in preparations of hypertensive animals, a condition associated with endothelial dysfunction (Félétou et al., 2009). Despite the presence of L-NAME, the inhibitory effect of the endothelium can be prevented by two NO scavengers and by a soluble guanylyl cyclase inhibitor. These findings permit us to conclude that the endothelium-derived factor present in the SHR aorta actually is NO.
Expression of eNOS is increased in young (around 12 weeks) SHR (Vaziri et al., 1998; Graham and Rush, 2004) and decreased in adult and old (around 36 and 72 weeks, respectively) SHR (Chou et al., 1998; Tang and Vanhoutte, 2008). Irrespective of the possible changes in the eNOS expression in hypertensive animals, the concentration of L-NAME (10−4 M) used in the present experiments indeed abrogated the activity of eNOS, as demonstrated by the abolition of relaxations to acetylcholine. Several other studies have examined expression of another NOS isoform, iNOS; in SHR, iNOS has been found to be up-regulated (Chou et al., 1998; Vaziri et al., 1998), undetectable (Khadour et al., 1997), expressed only upon stimulation (Boulanger et al., 1998), or expressed at levels similar to those observed in WKY (Nava et al., 1995). Thus, the contribution of iNOS to NO production in SHR remains unclear. However, the selective inhibitor of iNOS, 1400W, does not affect the endothelium-dependent inhibition of phenylephrine-induced contractions; this observation precludes the possibility that the inducible isoform of the enzyme contributes to the eNOS independent, endothelial NO-mediated inhibition of contraction. The neuronal isoform, nNOS, is expressed in the SHR aorta but only in the smooth muscle cells, and it needs to be activated by angiotensin II (Boulanger et al., 1998). Thus, under the present experimental conditions, the activity of NOS must be negligible, confirming that this enzyme(s) is not responsible for the observed NO-dependent inhibition due to the presence of endothelial cells. The fact that oxyhemoglobin, an extracellular scavenger of NO (Lancaster, 1997) prevents this inhibition implies that the NO involved transits through or is produced in the extracellular space of the vascular smooth muscle cells.
The present study demonstrates that the endothelium of hypertensive, but not normotensive, animals releases NOS-independent NO. This conclusion seems to contradict the current consensus (Zalba et al., 2001; Vanhoutte et al., 2005) that there is a reduced vasodilator influence by the endothelium when it becomes dysfunctional. The present findings may help to explain why earlier bioassay studies demonstrated that, in the presence of indomethacin, the release of EDRF/NO (EDNO) in the SHR aorta is not different from that in the WKY (Lüscher et al., 1988). Thus, the release of NOS-independent EDNO may be a compensatory mechanism of the endothelium to the reduced release of NO by eNOS in essential hypertension.
Compared with the WKY, the production of ROS upon activation of cyclooxygenase is higher in the SHR aorta (Tang et al., 2007). ROS contribute to endothelial dysfunction in part by reducing NO bioavailability and uncoupling eNOS (Wolin, 2000; Higashi et al., 2009). The NOS-independent, NO-dependent inhibitory effect of the presence of the endothelium was unmasked when cyclooxygenase was inhibited by indomethacin or meclofenamic acid. The suppression by NO can be seen only in the absence of ROS. Indeed, the prevention of the production of ROS obviously plays a key role in the appearance of the endothelium-dependent inhibition of the response to phenylephrine in the SHR aorta. This conclusion is based on the observation that the unmasking effect of indomethacin [which limits the production of ROS in this preparation by inhibition of cyclooxygenase (Tang et al., 2007)] is mimicked by apocynin, which acts mainly as a nonselective antioxidant at the concentration used in the present study (Brandes, 2010).
The present study confirms that, compared with its normotensive control, the WKY, the SHR has a higher concentration of nitrate/nitrite in both plasma and aorta (Alaghband-Zadeh et al., 1996; Wu and Yen, 1999). Nitrate and/or nitrite are the logical source of the NOS-independent NO, since they are the metabolic end-products of the NO-pathway that can be reduced to again form bioactive nitrogen oxides, including NO (Lundberg et al., 2008). Thus, hemoglobin, myoglobin, and xanthine oxidoreductase can transform nitrite to NO in various tissues and under different conditions (Chen et al., 2008; Lundberg, 2009). Cytochrome P450 reductase and cytochrome P450 can transform organic nitrate and nitrite to NO in vitro, respectively (Li et al., 2006). In the present study, DPI but not CLT (cytochrome P450 inhibitor) abolished the difference in phenylephrine-induced contractions between SHR aortae with and without endothelium. DPI is a flavoprotein inhibitor that can bind to the flavin site of several enzymes. Among those, DPI has been shown convincingly to inhibit cytochrome P450 reductase (McGuire et al., 1998; Ratz et al., 1999; Zhukov and Ingelman-Sundberg, 1999; Ratz et al., 2000; Day and Kariya, 2005; Li et al., 2006; Portal et al., 2008). However, DPI also can inhibit NADPH oxidases (Griendling et al., 1994), xanthine oxidoreductase (O'Donnell et al., 1993), nitric oxide synthases (Stuehr et al., 1991), and mitochondrial respiration [Complex I (Majander et al., 1994)], all of which could impact on the responses measured in the present study. Different inhibitors of those other enzymes were used to determine whether they would affect the NOS-independent NO release: 1) NADPH oxidase: apocycin, used at a concentration that should inhibit NADPH oxidase besides exerting nonspecific antioxidant properties (Brandes, 2010), decreased rather than increased the contractions evoked by phenylephrine; 2) xanthine oxidoreductase: allopurinol, an inhibitor of xanthine oxidoreductase (Puig et al., 1989) did not reverse the inhibitory effect of the endothelium; 3) nitric oxide synthases: the L-NAME and 1400W experiments already demonstrated that the phenomenon is NOS independent; and 4) mitochondrial respiration (Complex I): Complex III (not Complex I) can transfer nitrite to NO (Kozlov et al., 1999; Nohl et al., 2000), but only in an anoxic environment (Kozlov et al., 1999; Nohl et al., 2000; van Faassen et al., 2009). In the present study, the organ chambers were bubbled with 95% O2, which can hardly be considered as an anaerobic condition. In addition, inhibition of Complex I only partly reduces NO release, whereas inhibition of Complex III completely abolishes its release (Kozlov et al., 1999). An inhibitor of another enzyme that is a source of NO, aldehyde dehydrogenase 2 (ALDH-2) (Chen et al., 2002; Beretta et al., 2012), was also examined. Daidzin, used at a concentration reported to inhibit ALDH-2 (Keung et al., 1997), did not affect the inhibition by the endothelium of the contraction to phenylephrine.
Taken in conjunction, those findings support the interpretation that 1) the inhibitory effect of DPI observed in the present study can be attributed to inhibition of cytochrome P450 reductase, and 2) the NOS-independent NO is formed from endogenous nitrate by the action of cytochrome P450 reductase in the endothelium of the SHR aorta. As exogenous nitrate only induced minimal endothelium-dependent relaxation, it needs to be converted to NO to cause significant inhibition of phenylephrine-induced contraction. While nitrite is not the source of this NOS-independent EDNO, it may still play a role in vascular relaxation. Low concentrations of endogenous nitrite may lead to the release of undetectable amounts of NO from the endothelium. This possibility is suggested by the experiments on cultured HAECs. Indeed, the cells converted both exogenous nitrate and nitrite into NO, and these actions were prevented by DPI and CLT, respectively.
As the production of endothelium-derived NOS-independent NO is present only in the SHR aorta, hypertension may be associated with biochemical or genomic changes that promote NO formation from nitrate. The present findings demonstrate that an increased protein expression of cytochrome P450 reductase does not explain the differences between SHR and WKY aortae. Since the total nitrate and nitrite level is higher in the plasma and aorta of the hypertensive strain, the greater release of NOS-independent NO may be due to increased activity of the enzyme or to a higher concentration of substrate (nitrate and nitrite).
Modest dietary intake of inorganic nitrate reduces arterial blood pressure, inhibits platelet function, and prevents endothelial dysfunction in humans (Lundberg et al., 2011; Machha and Schechter, 2011; Pattillo et al., 2011). The findings of the present suggest the existence of an endogenous development of a nitrate-using mechanism in the SHR that may combat endothelial dysfunction.
Participated in research design: Zhao, Leung, Vanhoutte.
Conducted experiments: Zhao.
Performed data analysis: Zhao.
Wrote or contributed to the writing of the manuscript: Zhao, Leung, Vanhoutte.
The authors current research is supported by the Hong Kong Research Grant Council (University of Hong Kong, NO.769808M); Research Centre of Heart, Brain, Hormone & Healthy Aging of the University of Hong Kong; and the World Class University program (R31-20029) funded by the Ministry of Education, Science and Technology, South Korea.
Part of this work was presented previously as a poster: Zhao Y, Vanhoutte PM and Leung SWS (2012) Endothelial NOS-independent release of nitric oxide in the aorta of the spontaneously hypertensive rat, at the Experimental Biology 2012 Conference; 2012 April 21–25; San Diego, CA.
- N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride
- dimethyl sulfoxide
- endothelium-derived relaxing factor
- endothelial nitric oxide synthase
- human aortic endothelial cells
- inducible nitric oxide synthase
- Nω-nitro-L-arginine methyl ester
- nitric oxide synthase
- neuronal nitric oxide synthase
- prostaglandin E2
- reactive oxygen species
- spontaneously hypertensive rats
- Wistar Kyoto rats
- Received July 25, 2012.
- Accepted August 30, 2012.
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics