Higher Level of Plasma Nitric Oxide in Spontaneously Hypertensive Rats

AJH 1999;12:476–482

Chin-Chen Wu and Mao-Hsiung Yen Downloaded from https://academic.oup.com/ajh/article/12/5/476/181114 by guest on 30 September 2021

We had detected a slightly, but signiﬁcantly, SHR treated with LPS for 3 h. In vitro, the ACh- higher level of plasma nitrite/nitrate in the induced relaxation was attenuated in the aortae sponstanously hypertensive rat (SHR) by using the obtained from SHR. However, this difference nitric oxide (NO) analyzer (Sievers 280 NOA), between SHR and WKY (without LPS treatment) which converts nitrate (including nitrate converted was abolished by treatment of rings with L-NAME from nitrite) to NO. Here, we examined whether (30 ␮mol/L), suggesting that an impairment of NO the release of NO from protein-bound dinitrosyl formation was observed in the SHR. After rats nonheme iron complexes (DNIC) contributes to the were treated with LPS for 3 h, the ACh-induced elevated plasma nitrate level in the SHR. The SHR relaxation was reduced in the WKY, but not in the and their genetic normotensive controls, Wistar- SHR. In addition, a 10-fold increase of L-NAME Kyoto rats (WKY), were anesthestized and was needed to abolish the difference in ACh- cannulized for monitoring blood pressure, induced relaxation between SHR and WKY, collecting a blood sample, and the administration indicating an expression of inducible NO synthase of endotoxin (lipopolysaccharide [LPS]). The in both strains treated with LPS. We suggest that nitrate levels (an indicator of NO formation) in the the elevated plasma NO level in SHR may be due plasma and the aorta were measured by an NO to the release of NO from DNIC in the vascular analyzer. In addition, the relaxation of bed to combat the hypertensive state. Am J acetylcholine (ACh) in the presence or absence of Hypertens 1999;12:476–482 © 1999 American ␻ N -nitro-L-arginine methyl ester (L-NAME) was Journal of Hypertension, Ltd. also examined in thoracic aortae obtained from both strains. The slight, but signiﬁcant, increase of ␻ basal nitrate levels in the plasma and aorta were KEY WORDS: Nitric oxide, N -nitro-L-arginine methyl observed, and the former was further enhanced in ester, spontaneously hypertensive rats.

he discovery by Furchgott and Zawadzki in ically equivalent to nitric oxide (NO)2,3 or a biochem- 1980 of endothelium-derived relaxing factor ical congener thereof.4 NO is produced by the conver- (EDRF)1 has now been proved to play a cen- sion of the terminal guanidine nitrogen atom(s) from tral role in the cardiovascular system. A few l-arginine into l-citrulline, or a complex molecule Tyears later, the EDRF had been identiﬁed to be chem- yielding NO.5,6 The importance of NO in the physio-

Received April 2, 1998. Accepted November 11, 1998. Parts of this study had been presented in the 17th Scientiﬁc From the Department of Pharmacology, National Defense Medi- Meeting of the International Society of Hypertension. cal Center, Taipei, Republic of China, Taiwan. Address correspondence and reprint requests to Dr. Mao-Hsiung This work was supported by grants NSC 87-2314-B-016-058-M37 Yen, Department of Pharmacology, National Defense Medical Cen- (C. C. Wu) and NSC 87-2314-B-016-059-M37 (M. H. Yen) from the ter, P.O. Box 90048-504, Taipei, Republic of China, Taiwan. National Science Council, Republic of China, Taiwan.

logic control of blood pressure is now well estab- protein-bound dinitrosyl nonheme iron complexes lished. In addition, NO also prevents the activation (DNIC) in blood vessels. Therefore, the aim of this and adhesion of platelets and neutrophils to the en- study was to delineate the effect of NO in hyperten- dothelium, dilates vascular smooth muscle, and inhib- sion and to evaluate whether the release of NO from its cell mitogenesis and proliferation.7,8 NO causes DNIC contributes to the higher plasma NO level in vascular smooth muscle relaxation by activation of SHR. In this study, we examined NO metabolite levels soluble guanylate cyclase and by increasing cyclic (ie, nitrite/nitrate) in the plasma and aorta by using guanosine 3Ј,5Ј-monophosphate (cGMP) levels,9,10 the NO analyzer. ϩ causing a decrease in intracellular Ca2 . Therefore, MATERIALS AND METHODS alterations in NO synthesis may be an important factor in the pathogenesis of hypertension. In Vivo Experiments Sixteen-week–old male SHR It has been shown that the generation of NO from and WKY rats, whose stock originated from the Downloaded from https://academic.oup.com/ajh/article/12/5/476/181114 by guest on 30 September 2021 l-arginine by NO synthase (NOS) is an important Charles River Breeding Laboratories in Japan, were autocrine and paracrine signaling pathway in the reg- purchased from the Deparment of Laboratory Animal 7 ulation of various cell functions. In the vascular wall, Science of the National Defense Medical Center. Sys- 2ϩ endothelial cells express a constitutive Ca -calmod- tolic blood pressure was measured in conscious rats 11 ulin–dependent NOS (NOS III). A second type of by the tail-cuff method using a MOD 59 tail sphyg- 2ϩ NOS (Ca -calmodulin–independent [NOS II]) can be mograph (Blood Pressure Meter/Amplifier, Itic Inc., expressed in smooth muscle cells and fibroblasts by Woodland Hills, CA) every week. The systolic blood ␤ cytokines such as interleukin-1 or tumor necrosis pressure of SHR and WKY rats was 189 Ϯ 11 mm Hg ␣ factor- , or by mechanical, viral, or bacterial inju- (n ϭ 36) and 144 Ϯ 7mmHg(nϭ 20), respectively. 7,12 ries. The NOS II induced by cytokines and endo- Animals were anesthetized by intraperitoneal injec- toxin (lipopolysaccharide [LPS]) produces larger tion of a combination of urethane (0.6 g/kg) and chlo- amounts of NO than NOS III does. ral hydrate (0.4 g/kg). The trachea was cannulated to There are numerous reports showing that endothe- facilitate respiration and rectal temperature was main- lium-dependent relaxations of isolated vascular rings tained at 37°C with a homeothermic blanket (Harvard from spontaneously hypertensive rats (SHR) are im- Apparatus Ltd., South Natick, MA). The right femoral paired when compared with those from Wistar-Kyoto artery was cannulated and connected to a pressure 13–15 (WKY) rats. These results suggest that dysfunc- transducer (P23ID, Statham, Oxnard, CA) for the mea- tion of the endothelium causes an abnormal relax- surement of phasic and mean arterial blood pressure 16,17 ation, or a simultaneous release of endothelium- (MAP) and heart rate, which were displayed on a dependent contracting factor(s) decreases the relaxing Grass model 7D polygraph recorder (Grass Instru- 17,18 activity of NO. Any decrease in NO generation ments, Quincy, MA). The left femoral vein was can- may have a substantial effect on blood pressure and nulated for the administration of Escherichia coli LPS (5 tissue blood flow. mg/kg). Please note that the injection of LPS in rats The NO Analyzer (Sievers 280 NOA, Sievers Inc., was further done to examine the effect of basal expres- Boulder, CO) has been developed for the direct assess- sion of iNOS in SHR on hemodynamic changes. Upon 19–21 ment of NO level in exhaled air and of NO me- completion of the surgical procedure, cardiovascular 22–24 tabolites in the liquid system (eg, plasma), and has parameters were allowed to stabilize for 20 min. After 25–28 been used extensively in recent years. This ma- recording baseline hemodynamic parameters, animals chine is more sensitive than the Griess assay and more received either saline (ie, sham-operated) or LPS and 29 stable than the electrode system, indicating that the were monitored for 3 h. NO Analyzer is reliable for the detection of NO and its metabolites. Determination of Levels of Plasma Nitrate Before Recently, a study has shown that an overproduction and at 1, 2, and 3 h after injection of LPS, 0.25 mL of of NO is involved in the interaction between macro- blood was collected from a catheter placed in the phages or vascular smooth muscle cells and lympho- femoral artery. Any blood withdrawn was immedi- cytes in SHR, suggesting a general activation of the ately replaced by intravenous (iv) injection of an equal NO synthesis system in SHR.30 In contrast to earlier amount of saline. The blood sample was centrifuged studies, Nava and colleagues have recently reported (7200 g for 3 min) to prepare plasma and plasma was that increased activity of NOS III is observed in car- kept in a Ϫ70°C freezer. At a later stage, plasma diac endothelium in SHR.31 In addition, our previous samples were thawed and deproteinized by incubat- studies also showed a basal expression of NOS II in ing them with 95% ethanol (4°C) for 30 min. These the aorta from SHR.32,33 A recent study demonstrated samples were subsequently centrifuged for 5 min at that NO can be generated independently of NOS.34 It 14,000 g. The nitrate concentration in plasma depicted proposed that NO reacts with nonheme iron to form in this study is actually the total nitrite and nitrate 478 WU AND YEN AJH–MAY 1999–VOL. 12, NO. 5

concentration in plasma. In this method nitrate is re- ation of ACh was also examined in the presence of the ␻ duced to NO via nitrite. The amounts of nitrate in the NOS inhibitor N -nitro-l-arginine methyl ester (L- plasma (2 ␮L) were measured by adding a reducing NAME, 30 to 300 ␮mol/L).

agent (0.8% VCl3 in 1 N HCl) to the purge vessel to convert nitrate to NO, which was stripped from the Chemicals Acetylcholine chloride, bacterial lipo- plasma using a helium purge gas. The NO was then polysaccharide (E. coli serotype 0127:B8), phenyleph- drawn into the Sievers Nitric Oxide Analyzer (Sievers rine HCl, L-NAME, N-acetyl-l-cysteine, sodium ni- 280 NOA, Sievers Inc., Boulder, CO). Nitrate concen- trate, and urethane were purchased from Sigma trations were calculated by comparison with standard Chemical Co. Vanadium chloride (VCl3) was obtained solutions of sodium nitrate (Sigma Chemical Co., St. from Aldrich Chemical Co. (Milwaukee, WI). Louis, MO). Statistical Analysis All values in the figures and text Downloaded from https://academic.oup.com/ajh/article/12/5/476/181114 by guest on 30 September 2021 Determination of Levels of Aortic Nitrate Thoracic are expressed as mean Ϯ SEM of n observations, aortae were obtained from SHR and WKY without where n represents the number of animals or experi- LPS treatment. Two rats’ aortae from each group were ments performed. Statistical evaluation was per- pooled together (ie, n ϭ 1) to measure the nitrate in the formed by using ANOVA followed by a multiple com- vascular smooth muscle. After they were cleaned of parison test (Scheffe´’s test), except for the aortic nitrate fat and connective tissues, aortae were frozen in liquid study, which was analyzed by unpaired Student’s t nitrogen and stored for no more than 2 weeks at test. A value of P Ͻ .05 was considered to be statis- Ϫ80°C before assay. At a later stage, frozen samples tically significant. were thawed and cut into pieces and these samples RESULTS were homogenized on ice with a polytron PT MR 3000 homogenizer (Kinematic AG, Littau) in a buffer com- Effects of LPS on Blood Pressure and Plasma Nitrate posed of (mmol/L): Tris-HCl 50, EDTA 0.1, EGTA 0.1, Although rats were anesthetized, the MAP was higher 2-mercaptoethanol 12, and phenylmethylsulphonyl in the SHR than in the WKY. The injection of saline fluoride 1 (pH 7.4). Aortic homogenates were then instead of LPS did not cause any significant changes of further incubated with N-acetyl-l-cysteine (an agent MAP during the experimental period (eg, WKY: 114 Ϯ that decomposes the dinitrosyl nonheme iron com- Ϯ ϭ Ͼ 34 3 mm Hg at time 0 v 108 5mmHgat3h,n 8, P plexes, 30 mmol/L) for 10 min. This reaction was .05; SHR: 154 Ϯ 8 mm Hg at time 0 v 144 Ϯ 8at3h, stopped with adding 1% trichloroacetic acid (TCA, n ϭ 12, P Ͼ .05) in the sham-operated groups (Figure 4°C) and subsequently these samples were centri- 1A, left panel). In contrast, the injection of LPS re- fuged for 7 min at 13,000 g. The amounts of nitrate in ␮ ␮ sulted in a fall of MAP after 1 h until the end of the the supernatant (30 L, approximately 60 g protein) experiment (ie, at 3 h after LPS) in both SHR and WKY were injected into the NO analyzer as described ear- (Figure 1A, right panel). The dose of LPS used in this lier. study caused only a mild hypotension (ϳ30 mm Hg Organ Bath Experiments At the end of the in vivo fall at 3 h) in the WKY rats; it had a much greater experiments, rats were exsanguinated and the thoracic hypotensive effect (ϳ61 mm Hg fall at 3 h) in the SHR. aorta was excised and placed in cold physiologic salt In other words, at 3 h after LPS the MAP values solution (PSS; 4°C). Control aortae were also obtained between WKY and SHR were not significantly differ- from untreated (ie, sham-operated) rats. Fat and con- ent from each other. In addition, four of 24 SHR nective tissues were trimmed from the aorta and aor- (which were not included in our MAP data) had a tae were then cut into rings (3 to 4 mm in width). severe hypotension and died before the experimental While preparing these rings, care was taken to avoid period, whereas none of the WKY rats exhibited this damaging the endothelium. The rings were left to effect. equilibrate for 1 h under an optimal resting tension of Figure 1B (left panel) shows that basal levels of 2.0 g (in the WKY group) or 1.8 g (in the SHR group) plasma nitrate were 12.3 Ϯ 1.3 and 7.4 Ϯ 0.4 ␮mol/L and washed every 15 min. Before the relaxation stud- in SHR and WKY, respectively, significantly higher in ies, rings were challenged twice with phenylephrine SHR than in WKY. In addition, the operation did not (PE, 0.3 ␮mol/L) to ensure the reproducibility of the cause a significant rise of plasma nitrate, as there were observed contractile response. Drugs were removed no significant changes during the experimental period from the bath by several washes with PSS, and the in both strains (Figure 1B, left panel). After treatment tension was allowed to return to baseline. Again, rings of rats with LPS, the increment of plasma nitrate was were contracted with PE (0.3 ␮mol/L). When maximal in a time-dependent manner in both strains (Figure stable contractile responses were reached, acetylcho- 1B, right panel). However, the ability of LPS to in- line (ACh, 1 nmol/L to 1 ␮mol/L) was added to the crease nitrite was greater in SHR than in WKY (ie, at bath in a cumulative manner. In addition, the relax- 1 h, twofold v 1.6-fold; at 3 h, 12.6-fold v 9.4-fold). AJH–MAY 1999–VOL. 12, NO. 5 NITRIC OXIDE IN SHR 479

FIGURE 1. (A) Mean arterial blood pressure (MAP) and (B) plasma nitrate levels measured in anesthetized WKY and SHR

treated with either saline (left Downloaded from https://academic.oup.com/ajh/article/12/5/476/181114 by guest on 30 September 2021 panel,nϭ 8 and n ϭ 12, respectively) or LPS (right panel,nϭ 12 and n ϭ 20, respectively) for 3 h. Values are means Ϯ SEM. WKY, Wistar-Kyoto rats; SHR, spontaneously hypertensive rats; LPS, E. coli lipopolysaccharide. *P Ͻ .05 v WKY. †P Ͻ .05 v time 0 in the same group.

Changes of Nitrate Levels in the Aorta To examine pendent relaxation in endothelium-intact aortic rings the basal level of nitrate in the blood vessel wall, the obtained from WKY and SHR (Figure 3, left panel). aorta was obtained from rats without LPS treatment. However, the relaxation caused by ACh was reduced Figure 2 shows that the basal level of aortic nitrate is in the SHR, compared with WKY. In the presence of 30 higher in SHR than in WKY. ␮mol/L L-NAME, the difference of ACh-induced relaxation between SHR and WKY was abolished. It is Effects of L-NAME on ACh-Induced Relaxations In noted that in rats treated with LPS, the dose-response the sham-operated groups, ACh evoked a dose-de- curve of ACh was shifted to the right in rings obtained from WKY rats, but not in those from SHR (Figure 3, left and right panels). Interestingly, at the same concentration of L-NAME (ie, 30 ␮mol/L), the difference in ACh-induced relaxation between SHR and WKY was not signiﬁcantly affected. However, this difference was abolished by increasing the concentration of L-NAME to 300 ␮mol/L (Figure 3, right panel). In this experiment, we observed that there were two prepa- rations from the SHR (in response to ACh) resistant to 300 ␮mol/L L-NAME, whereas they needed 1 ␮mol/L L-NAME to abolish the ACh relaxation (data not shown). DISCUSSION

FIGURE 2. Aortic nitrate levels detected in WKY (open col- We have demonstrated that the intravenous injection umn, n ϭ 4) and SHR (grey column, n ϭ 6) without LPS of LPS (5 mg/kg) caused a mild hypotension in nor- treatment. Values are means Ϯ SEM. WKY, Wistar-Kyoto rats; motensive rats, but induced a more severe hypoten- SHR, spontaneously hypertensive rats; LPS, E. coli lipopolysac- sive effect in SHR. Using treatment with LPS, the charide. *P Ͻ .05 v WKY. plasma levels of nitrate were signiﬁcantly increased in 480 WU AND YEN AJH–MAY 1999–VOL. 12, NO. 5

FIGURE 3. Effects of L-NAME (30 or 300 ␮mol/L) on ACh-induced relaxations in aortic rings obtained from WKY and SHR treated with either saline (left panel,nϭ 8 and n ϭ 12, respectively) or LPS (right panel,nϭ 12 and n ϭ 18, respectively) for 3 h. Values are means Ϯ SEM. WKY, Wistar-Kyoto rats; SHR, spontaneously hypertensive rats; LPS, E. coli lipopolysaccharide; L-NAME,

␻ Downloaded from https://academic.oup.com/ajh/article/12/5/476/181114 by guest on 30 September 2021 N -nitro-l-arginine methyl ester; ACh, acetylcholine. *P Ͻ .05 v WKY with the same treatment.

both strains and were significantly higher in SHR. Indeed, the production of NO in cultured SHR vascu- Interestingly, a slight, but significantly, higher basal lar smooth muscle cells was greater than that in level of plasma nitrate was observed in the SHR. This WKY.30,40 Here, we have also demonstrated that the higher basal level of plasma nitrate in SHR could be level of NO release from DNIC was higher in the aorta due to an increased activity of NOS III in the cardiac from SHR. This may contribute to a larger expression 31 endothelium or to a basal expression of NOS II in the of the soluble guanylate cyclase in the smooth muscle 32,33 aorta. Although we did not have good evidence from SHR than from WKY.41 showing that this difference is important from a bio- The treatment of rats with LPS did not further im- logic standpoint, a recent study has demonstrated that pair the ACh-induced relaxation in SHR ex vivo, but it NO function in the vascular beds can be enhanced in significantly attenuated that response in WKY. This is the hypertensive state and provides a compensatory consistent with previous studies showing that bacte- mechanism to keep the blood pressure and peripheral 35 rial LPS produces a selective diminution of the endo- resistance at lower levels. This was also supported thelium-dependent vasodilator actions in an E. coli by results showing that NO blockade produced an endotoxemia model.42 Because the endothelium-de- exaggerated pressor response in SHR compared with 36,37 pendent vasodilatation has been impaired in the SHR, WKY. the treatment of SHR with LPS may not be able to Based on the fact that the mechanism of NO-in- further reduce NOS III activity, as we demonstrated in duced relaxation in the aorta is not different from that our previous studies.33 Suprisingly, the treatment of in the resistant blood vessels at the level of the smooth aortic rings with the NOS inhibitor L-NAME (30 muscle cell, and that the production of NO contributes ␮mol/L, which completely inhibited ACh-induced re- mainly to the relaxation in large conduitory blood laxation in the sham-operated group) only partially vessels rather than in the resistance vessels,38,39 we shifted the dose-response curve of ACh to the right in therefore examined the effect of NO in the aorta instead of in the resistance vessels. Our in vitro studies both strains treated with LPS, indicating an expression manifest that an impaired relaxation to ACh was of NOS II in the endothelium. The NOS inhibitor L-NAME, at a lower concentration (Ͻ30 ␮mol/L), is found in aortic rings from SHR, as other studies re- 43 44 ported,16–18 suggesting that dysfunction of the endo- more selective for NOS III or neuronal NOS, Ͼ ␮ thelium causes an abnormal relaxation16,17 or that a whereas at a higher concentration ( 300 mol/L), it 43,44 simultaneous release of endothelium-dependent con- also inhibits NOS II activity. It seems that the tracting factor(s) decreases the relaxing activity of expression of endothelial NOS II, which is more resis- NO.17,18 Here, we show that the difference caused by tant to the NOS inhibitor L-NAME, is inhibited by a ACh in both strains was abolished by the NOS inhib- higher concentration of L-NAME, as it has abolished itor L-NAME, suggesting that this difference between the ACh-induced relaxation. Although there are no WKY and SHR comes from NO. This result argues studies showing that endothelial NOS II is responsible strongly against the possibility that NOS III activity is for the relaxation caused by ACh, it appears to be a increased in the aortic endothelium, as reported in the possible mechanism for this interpretation. Further- cardiac endothelium of SHR.31 Thus, it is more likely more, the difference of ACh-induced relaxation be- that a higher basal plasma nitrate level is due to a tween WKY and SHR caused by LPS may also be due basal expression of NOS II in the aorta from SHR. to the synthesis of NO (presumably via NOS II), as the AJH–MAY 1999–VOL. 12, NO. 5 NITRIC OXIDE IN SHR 481

NOS inhibitor L-NAME (at a higher concentration) modulin-dependent endothelium-derived relaxing fac- could abolish that difference between WKY and SHR. tor/nitric oxide synthase activity is present in the par- Although this study is largely confirmatory, it ticulate and cytosolic fractions of bovine aortic endothelial cells. Proc Natl Acad Sci USA 1991;88:1788– would be interesting to investigate possible changes in 1792. signal tranduction and molecular regulation of NOS in 12. Busse R, Mulsch A: Induction of nitric oxide synthase hypertension in future studies using biochemical or by cytokines in vascular smooth muscle. FEBS Lett molecular biologic techniques. For instance, Scott and 1990;275:87–90. colleagues have suggested that a decreased activity of 13. Watt PA, Thurston H: Endothelium-dependent relax- NOS III is linked to an increased activity of NOS II in ation in resistance vessels from the spontaneously hy- LPS-incubated tissues.45 In addition, we have pro- pertensive rats. J Hypertens 1989;7:661–666. posed a compensatory relationship between NOS III 14. Lee L, Webb RC: Endothelium-dependent relaxation and NOS II in the development of hypertension in and l-arginine metabolism in genetic hypertension. Downloaded from https://academic.oup.com/ajh/article/12/5/476/181114 by guest on 30 September 2021 SHR.33 Thus, we conclude that the enhanced basal Hypertension 1992;19:435–441. formation of NO may be attributed to the release of 15. Luscher TF, Dohi Y, Tschudi M: Endothelium-depen- NO from DNIC in the SHR, which then provides a dent regulation of resistance arteries: alterations with compensatory effect to combat the hypertensive state. aging and hypertension. J Cardiovasc Pharmacol 1992; 19(suppl):S34–S42. ACKNOWLEDGMENTS 16. Clozel M, Kuhn H, Hefti F, et al: Endothelial dysfunction and subendothelial monocyte macrophages in hy- We gratefully acknowledge Ms. M. H. Liao for her technical pertension. 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