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Regional Hemodynamics after Chronic Nitric Oxide Inhibition In Spontaneously Hypertensive Rats
Regional Hemodynamics after Chronic Nitric Oxide Inhibition In Spontaneously Hypertensive Rats JASMINA VARAGIC, MD, PHD; MIRJANA JERKIC, MD, PHD; DJURDJICA JOVOVIC, MD, PHD; DANICA NASTIC-MIRIC, PHD; GORDANA ADANJA-GRUJIC, MSc; JASMINA MARKOVIC-LlPKOVSKI, MD, PHD; VESNA LACKOVIC, MD, PHD; GORDANA RADUJKOVIC-KUBUROVIC, DDM, PHD; DUSAN KENTERA, MD, PHD
ABSTRACT: Background: Inhibition of nitric oxide (NO) synthase by L-arginine analogs is associated with elevation of blood pressure in rats. Because endotheliumdependent vasomotion in different vascular beds is not homogenous, the aim of this study was to characterize and compare regional hemodynamic responses in carotid, femoral, and renal vascular beds after chronic NO inhibition in spontaneously hypertensive rats. The possible role of circulating endothelin and renin angiotensin systems in mediating the effects of chronic NO inhibition was also studied. Methods: Systemic and regional hemodynamics, left ventricular mass, plasma renin activity, and plasma endothelin-1 were determined in control and NW-nitro-Larginine methyl ester (L-NAME)-treated (10 mglkglday, 4 weeks) spontaneously hypertensive rats. Results: L-NAME treatment increased arterial pressure and total peripheral and regional vascular resistance and decreased cardiac output, stroke volume, and regional blood flow. An in-
E
ndothelium is the major source of the endothelium-derived relaxing factor nitric oxide (NO), which affects contractile function 1 and proliferation and migration of vascular smooth muscle cells. 2 NO is derived from L-arginine in a process that can be competitively inhibited by such L-arginine analogs as NW-nitro-L-arginine methyl ester (L-NAME),1 Inhibition of NO synthesis causes vasoconstriction,3
From the Institute for Medical Research (JV, MJ, DJ, JM-L, VL, Medicine-Clinical Center
GR-I(, DK) and the Institute of Nuclear (DN-M, GA-G), Belgrade, Yugoslavia.
Submitted November 11, 1999; accepted in revised form February 15, 2000. This work was supported by a grant from the Ministry of Science and Technology of Serbia. Correspondence: Jasmina Varagic M.D., Ph.D., Hypertension Research, Alton Ochsner Medical Foundation, 1520 Jefferson Highway, New Orleans, LA 70121. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
crease in blood flow ratio and a decrease in vascular resistance ratio between carotid and renal as well as femoral and renal vascular beds in rats treated with L-NAME was found. Blood flow and vascular resistance ratios between femoral and carotid vascular beds remained unchanged. L-NAME increased plasma renin activity and left ventricular weightlbody weight ratio, whereas plasma endothelin-1 was not modified. Conclusions: The results of this study showed that the renal circulation seemed to be more sensitive to the effects of chronic NO inhibition than carotid and femoral vascular beds. Simultaneous activation of the renin angiotensin system may further potentiate cardiovascular effects of chronic NO inhibition. No evidence that circulating endothelin-1 plays a role in this model of hypertension was found. KEY INDEXING TERMS: Nitric oxide; Regional hemodynamics; Plasma renin activity; Endothelin; Spontaneously hypertensive rats. [Am J Med Sci 2000;320(3):171-6.]
increase in total peripheral resistance, and impairment in regional blood flOWS. 4- 7 Previous studies have demonstrated that NO has an important role in long-term regulation of arterial blood pressure in spontaneously hypertensive rats (SHR).8,9 It has been shown that chronic administration of the L-arginine analog L-NAME in SHR induced a significant rise in blood pressure accompanied with decreased cardiac output and increased total peripheral resistance. The effects of chronic NO synthase inhibition on renal9,10 and coronary circulationl l have also been studied. However, the hemodynamic response of other vascular beds to the chronic administration of L-NAME has not been examined in SHR. Because endothelium-dependent vasomotion in different vascular beds is not homogenous,12-14 this study was designed to characterize and compare regional hemodynamic responses in carotid, femoral, and renal vascular beds after chronic NO synthesis inhibition in SHR. Further171
Nitric Oxide Blockade in Spontaneously Hypertensive Rats
more, the attempt was made to delineate the possible role of circulating endothelin and renin-angiotensin system in mediating the effects of chronic NO synthesis inhibition.
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Material and Methods The protocol of the present study was approved by the Institutional Committee on Animal Care and Use of Laboratory Animals of Institute for Medical Research, Belgrade, Yugoslavia. Experimental Groups. Experiments were performed in adult male SHR weighing about 250 g. The rats were divided into 2 experimental groups: 1 group was given L-NAME in drinking water (average dose, 10 mg/kg/day; fresh solutions prepared daily) for 4 weeks; rats in the control group were given tap water. There were 24 rats in each group. All animals were given standard food for laboratory rats (Veterinarski zavod, Zemun, Yugoslavia). Experimental Protocol. Two separate baseline determinations of body weight and systolic blood pressure (tail-cuID were made in a span of 2 weeks in all rats. Afterward, animals were assigned randomly to their respective treatments for a period of 4 weeks. Measurements of blood pressure were made once a week during the 4-week course of the treatment. At the end of the fourth week, hemodynamic studies were done in 16 rats selected randomly from each group. In the remaining rats from each group, measurements of plasma endothelin-1 (ET-1) and plasma renin activity (PRA) were performed. At the end of the experiment, all rats were exsanguinated and their hearts and left kidneys removed. The atria were excised, the lateral right ventricular wall was separated from the left ventricle and septum, and their weights were determined on an automatic analytical balance. The ratio ofthe left and right ventricular weights as well as of the left kidney weight to body weight was determined. Experimental Techniques. Indirect determinations of systolic blood pressure were made using a tail-cuff pneumatic pulse detector and a recorder (Narco Bio-System, Houston, TX). For hemodynamic studies, rats were anesthetized with pentobarbital (35 mg/kg, intraperitoneal injection) and a tracheal cannula was inserted. Blood pressure was measured directly through a femoral artery catheter PE-50 with a low-volume displacement transducer P23Db and a direct writing recorder (Physiograph Four; Narco Bio-System). Mean arterial pressure (MAP) was obtained by electronic integration. Cardiac output (CO) was determined by a previously described15 modification of Coleman's16 application of the dye dilution technique. Indocyanine green was used as the indicator, and a recording densitometer (Beckman Instruments, Columbia, MD) was used for the determination of dye in the blood and registration of the dilution curve. Total peripheral resistance (TPR) was calculated from the MAP and CO (assuming that the mean right atrial pressure is zero) and is expressed as millimeters of Hg per milliliter per minute per 100 g of body weight. Stroke volume was calculated by dividing CO by heart rate. For blood flow measurement, femoral, carotid and renal arteries were carefully isolated. An ultrasonic flow probe (lRB, internal dimeter, 1 mm) was placed around the arteries for the measurement of total femoral (FBF), carotid (CBF), and renal blood flow (RBF) using a Transonic T106 Small Animal Bloodflow meter (Transonic Systems, Ithaca, NY). Regional blood flow was normalized to flow in milliliter per minute per 100 g of body weight. Regional vascular resistance (VR) was calculated by dividing the MAP by the regional blood flow and is expressed as millimeters of Hg per milliliter per minute per 100 g of body weight. From the blood samples collected from the abdominal aorta, hematocrit (Hct), and plasma creatinine concentrations were determined using the micromethod and Jaffe reaction. For determinations of plasma ET-1 and PRA, the animals were decapitated and blood samples were collected into chilled, siliconized centrifuge tubes containing Na-EDTA (1 mg/mL) and aprotinin (1000 kIU/mL) for ET-1 and Na-EDTA (2 mg/mL) for PRA. RPA 5359 Endothelin-1,2 biotrack assay system (provided
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by Amersham, Paisley, UK) and REN-CT2 angiotensin I radioimmunoassay kit (provided by CIS Bio International, Gif-surYvette, France) were used in radioimmunoassay procedures. Statistical Analysis. Data are presented as mean ± SEM. Comparisons among groups were made by Student's t-test. A level of P < 0.05 was considered significant.
Results
L-NAME treatment had no influence on body weight. Mean body weight at the end of experiment was 290 ± 6 g in control rats and 277 ± 7 g in L-NAME group. Changes in the systolic blood pressure during the 4 weeks oftreatment are shown in Figure 1. Systolic blood pressure rose progressively in L-NAMEtreated rats, reaching 232 ± 3 mm Hg by the end of the treatment, compared with 179 ± 2 mm Hg in untreated control rats. Mter the 4-week treatment, L-NAME significantly increased MAP. This elevation of arterial pressure in rats drinking L-NAME was accompanied by a significantly higher TPR and a substantial reduction in CO (Figure 2). No change in heart rate (368 ± 4 in control rats versus 365 ± 15 bpm in L-NAME-treated SHR) and Het (48 ± 2 in control rats versus 49 ± 3% in L-NAME-treated SHR) was found, whereas stroke volume decreased significantly in SHR given L-NAME (0.070 ± 0.007 in control rats versus 0.047 ± 0.005 mUbeatl100 gin L-NAME-treated SHR; P < 0.05). In rats drinking L-NAME, blood flow was significantly reduced and VR was increased in all vascular beds studied compared with the control rats (Figures 3, 4, and 5). Blood flow and VR ratios between tissue regions within the control and L-NAME group are shown in Fig 6. Intergroup evaluation revealed an increase in blood flow ratio and a decrease in VR ratio between carotid and renal as well as femoral and renal vasSeptember 2000 Volume 320 Number 3
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Figure 5. Changes in renal vascular bed in response to chronic NO synthesis inhibition; ***, P < 0.001; n=16 for each group. RVR, renal vascular resistance.
cular beds in rats treated with L-NAME. On the other hand, blood flow and VR ratio between femoral and carotid vascular beds remained unchanged after prolonged NO synthesis inhibition. These results
showed that vasoconstriction was more pronounced in renal vasculature than in the other vascular beds studied. Plasma creatinine level was higher after 4-week treatment with L-NAME (78.90 ± 3.61 in
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173
Nitric Oxide Blockade in Spontaneously Hypertensive Rats
Table 1. Organ Weights in SHR Given L-NAME
LVW (mg/100 g) RVW (mg/100 g) LKW (mg/100 g)
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314.55 ± 4.29 67.44 ± 2.26 340.03 ± 7.03
331.38 ± 6.38* 62.32 ± 2.76 337.17 ± 18.40
L VW, left ventricular weight/body weight ratio; RVW, right ventricular weight/body weight ratio; LKW, left kidney weight/body weight ratio. Values are means ± SEM; n = 24 for each group; *p < 0.05.
control versus 112.22 ± 12.27 mmollL in L-NAMEtreated rats; P = 0.057), although the difference between groups was not statistically significant. Chronic oral administration of L-NAME induced increase in PRA (1.6 ± 0.2 in control rats versus 24.65 ± 2.2 ng of angiotensin 1/mI1hr in L-NAMEtreated rats, P < 0.001), whereas plasma ET-1 was not modified by L-NAME treatment (27.2 ± 2.4 in control rats versus 21.6 ± 3.6 fmollmL in L-NAMEtreated rats). Left ventricular weightlbody weight ratio was found to be significantly increased in L-NAMEtreated rats, whereas right ventricular weightlbody weight ratio and left kidney weightlbody weight ratio of rats given L-NAME were not different from those in control SHR (Table 1). However, no correlation between PRA and left ventricular weightlbody weight ratio was found (r = 0.306). Discussion
The present results show that after treatment with L-NAME, VR is increased and blood flow decreased in all vascular beds studied. Furthermore, vasoconstriction was more pronounced in renal vasculature than in 2 other vascular beds studied, as indicated by a decrease in carotid VRfrenal VR and femoral VRfrenal VR ratios and an increase in CBFI RBF and FBFIRBF ratios. Therefore, our results suggest that inhibition of NO-mediated mechanisms contribute differently to the vascular tone in different vascular beds. Several in vitro and in vivo studies have shown that L-NAME increased sensitivity of renal arterioles to the vasoconstrictor effect of angiotensin 11.17,18 It seems likely that the combination of decreased NO synthesis with activated reninangiotensin system made renal circulation the most sensitive among the 3 vascular beds studied. A higher plasma creatinine concentration revealed decreased glomerular filtration rate that was also reported in few previous studies. 9,10 Carotid and femoral circulation displayed similar sensitivity to chronic NO synthesis inhibition. Prolonged inhibition of NO synthesis resulted in a severe elevation of arterial pressure and was associated with an increase in total peripheral resistance 174
and a decrease in cardiac output, suggesting that NO has an important role in long term control of arterial pressure in SHR. These results are in agreement with earlier reports. 8,9,19 Moreno et al. suggested that impairment of CO might be a consequence of a decreased contractility caused by myocardial ischemia. An increased work demand and a decreased coronary flow may lead to ischemia and impaired contractility in this model ofhypertension.19 On the other hand, in an acute study, inhibition of NO synthesis reduced coronary flow but did not affect myocardial oxygen consumption. 20 It should also be mentioned that after prolonged inhibition of NO synthesis, no signs of heart failure (weakness, pulmonary edema, hepatomegaly, ascites) were reported. It is possible that vasoconstriction induced by chronic NO synthesis inhibition does not result solely from removal of basal NO release but also from activation of vasoconstrictors; it has been shown that NO donors reduce basal and stimulated ET production. 21 Therefore, it may be expected that inhibition of NO synthesis would stimulate ET production. However, our results show that plasma ET-1 levels were not affected significantly by LNAME, indicating that circulating ET-1 does not have a role in progressive rise of blood pressure and the development of left ventricular hypertrophy in this model. In the other 2 studies, carried out in SHR, 3 weeks' treatment with L-NAME induced a slight increase in circulating ET.22,23 The differences between our results and those of other studies may be attributable to the differences in doses of LNAME. Furthermore, it was demonstrated that blood pressure elevation in SHR treated with LNAME is endothelin independent, because no response to chronic endothelin receptor blockade was observed. 23 Thus, it does not seem likely that ET plays a role in L-NAME-induced hypertension. The present results also showed that chronic NO synthesis inhibition in SHR increased PRA, which may further aggravate hypertension and ventricular hypertrophy. The data on the effect of NO synthesis inhibition on PRA are contradictory24-28; it may be suggested that renin secretion increases only after prolonged blockade of NO synthesis, which is associated with development of severe hypertension and vascular lesions in the kidneys. This has been previously described in normotensive rats given high doses of L-NAME28 and in SHR treated with 20 mg/kg/day of L-NAME.29 In the present study, a significant increase in left ventricular mass was also found in rats given LNAME. The reports concerning the development of left ventricular hypertrophy in experimental model of chronic NO synthesis inhibition8- 11 ,22,23,29,30 are conflicting. A number of studies show that in rats treated with inhibitors of NO synthesis, a significant increase in arterial pressure occurs, but left ventricSeptember 2000 Volume 320 Number 3
Vorogic at 01
ular hypertrophy develops only in animals with increased PRA.8,28-30 In contrast, no increase in left ventricular mass in L-NAME-treated SHR despite increased PRA has also been reported. 22 ,23 In our study, PRA was increased (range, 7.25-36 ng of angiotensin l/mUhr), but intragroup evaluation did not reveal any correlation between PRA and left ventricular mass in the L-NAME group. It is possible that 3 weeks ofL-NAME treatment was not long enough to produce a more prominent change in left ventricular mass, as noted by Moreno et al. 19 They found that the increase in left ventricular weight index in the L-NAME group was significant after 8 weeks but not in rats studied after 2 or 4 weeks of treatment. Indeed, only 5% increase in left ventricular mass in L-NAME group was found in our study. On the other hand, intense myocardial alteration (myocardial necrosis and fibrosis), frequently reported early in the course ofL-NAME treatment,19,31 may also influence left ventricular mass. The extent and duration of NO synthesis inhibition may be one of the factors that determined the development of left ventricular hypertrophy in this experimental model. All these factors complicated the interpretation of the data, but our results of significant differences in left ventricular weightlbody weight ratio and PRA between the control and L-NAME groups seem to support the thesis that the renin angiotensin system plays an important role in the pathogenesis of the cardiac hypertrophy in this experimental model. In summary, the results of this study showed that NO has an important role in the long-term regulation of arterial blood pressure and regional blood flow. It seems likely that simultaneous activation of the renin angiotensin system further potentiated cardiovascular effects of chronic NO inhibition. Compared with carotid and femoral vascular beds, renal circulation seemed to be more sensitive to the effects of chronic NO inhibition.
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Acknowledgements:
18.
The expert technical assistance of Mrs. Zaga Jovanovic is acknowledged. We thank Dr. Dinko Susic for helpful comments and suggestions.
19.
References
20.
1. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991;43:109-42. 2. Dubey RK, Luscher TF. Nitric oxide inhibits angiotensin II-induced migration of vascular smooth muscle cells [abstractJ. Hypertension 1993;22:412. 3. Rees DD, Palmer RMJ, Hodgson HF, et aI. A specific inhibitor of nitric oxide formation from L-arginine attenuates endothelium-dependent relaxation. Br J Pharmacol 1989;96: 418-24. 4. Meyer P, Flammer J, Luscher TF. Endothelium-dependent regulation of the ophthalmic microcirculation in the THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
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perfused eye: role of nitric oxide and endothelins. Invest Ophthalmol Vis Sci 1993;34:3614-21. Rees DD, Palmer RMJ, Hadson HF, et aI. Role of endothelium-derived nitric oxide in the regulation of blood pressure. Proc Natl Acad Sci USA 1989;86:3375-78. Baylis C, Mitruka B, Deng A. Chronic blockade of nitric oxide synthesis in the rat produces systemic hypertension and glomerular damage. J Clin Invest 1992;90:278-81. Gardiner SM, Compton AM, Kemp PA, et aI. Regional haemodynamic changes during oral ingestion of NG-monomethyl-L-arginine or NG-nitro-L-arginine methyl ester in conscious Brattleboro rats. Br J Pharmacol 1990;101:10-2. ArnaI JF, Battle T, Menard J, et aI. The vasodilatory effect of endogenous nitric oxide is a major counter-regulatory mechanism in the spontaneously hypertensive rat. J Hypertens 1993;11:945-50. Ono H, Ono Y, Frohlich DE. Nitric oxide synthase inhibition in spontaneously hypertensive rats. Systemic, renal, and glomerular hemodynamics. Hypertension 1995;26:249-55. Francischetti A, Ono H, Frohlich DE. Renoprotective effects of felodipine and/or enalapril in spontaneously hypertensive rats with and without L-NAME. Hypertension 1998; 31:795-801. Fujita H, Takeda K, Nakamura K, et aI. Role of nitric oxide in impaired coronary circulation and improvement by angiotensin II receptor antagonist in spontaneously hypertensive rats. Clin Exp Pharmacol Physiol 1995;22:S148-50. Dohi Y, Thiel MA, Buhler FR, et aI. Activation of the endothelial L-arginine pathway in pressurized mesenteric resistance arteries: effect of age and hypertension. Hypertension 1990;15:170-9. Luscher TF. Endothelium-derived nitric oxide: the endogenous nitrovasodilatator in the human cardiovascular system. Eur Heart J 1991;12 Suppl E:2-11. Tschudi MR, Cuiscione L, Luscher TF. Effect of aging and hypertension on endothelial function of rat coronary arteries. J Hypertens 1991;9:164-5. Susic D, Haxhiu M, Kentera D. Decreased pulmonary pressor response to acute hypoxia in chronically hypoxic rats. Respiration 1981;41:166-73. Coleman TG. Cardiac output by dye dilution in the conscious rat. J Appl PhysioI1974;37:452-4. Ito S, Johnson JS, Carretero OA. Modulation of angiotensin II-induced vasoconstriction by endothelium-derived relaxing factor in the isolated microperfused rabbit afferent arteriole. J Clin Invest 1991;87:1656-63. Alberola AA, SaIazar FJ, Nakamura T, et aI. Interaction between angiotensin II and nitric oxide in control of renal hemodynamics in conscious dogs. Am J Physiol 1994;267: R1472-8. Moreno H Jr, Metze K, Bento AC, et aI. Chronic nitric oxide inhibition as a model of hypertensive heart muscle disease. Basic Res Cardiol 1996;91:248-55. Mankad P, Yacoub M. Influence of basal release of nitric oxide on systolic and diastolic function of both ventricles. J Thorac Cardiovasc Sur 1997;113:770-6. Saijonmaa 0, Ristimiiki A, Fyhrquist F. Atrial natriuretic peptide, nitroglycerine, and nitroprusside reduce basal and stimulated endothelin production from cultured endothelial cells. Biochem Biophys Res Commun 1990;173:514-20. Sventek P, Li JC, Grove K, et aI. Vascular structure and expression of endothelin-1 gene in L-NAME treated spontaneously hypertensive rats. Hypertension 1996;7:49-55. Li J-S, Deng LY, Grove K, et aI. Comparison of effect of endothelin antagonism and angiotensin-converting enzyme inhibition on blood pressure and vascular structure in spon-
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taneously hypertensive rats treated with NW-nitro-L-arginine methyl ester. Correlation with topography of vascular endothelin-1 gene expression. Hypertension 1996;28:188-95. Dananberg J, Sider RS, Grekin RJ. Sustained hypertension induced by orally administered nitro-L-arginine. Hypertension 1993;21:359-63. Johnson RA, Freeman RH. Renin release in rats during blockade of nitric oxide synthesis. Am J Physiol 1994;266:R1723-9. Johnson RA, Freeman RH. Sustained hypertension in the rat induced by chronic blockade of nitric oxide production. Am J Hypertens 1992;5:919-22. Manning RD, Hu L, Mizelle HL, et at. Role of nitric oxide in long-term angiotensin II-induced renal vasoconstriction. Hypertension 1993;21:949-55.
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28. Zanchi A, Schaad N, Osterheld M-C, et at. Effects of chronic NO synthase inhibition in rats on renin-angiotensin system and sympathetic nervous system. Am J Physiol 1995; 268:H2267-73. 29. Vaskonen T, Mervaala E, Krogerus L, et al. Cardiovascular effects of chronic inhibition of nitric oxide synthesis and dietary salt in spontaneously hypertensive rats. Hypertens Res 1997;20:183-92. 30. Arnal JF, EI Amrani AI, Chatellier G, et at. Cardiac weight in hypertension induced by nitric oxide synthase blockade. Hypertension 1993;22:380-7. 31. Numaguchi K, Egashira K, Takemoto M, et al. Chronic inhibition of nitric oxide synthesis causes coronary microvascular remodeling in rats. Hypertension 1995;26:957-62.
September 2000 Volume 320 Number 3
ABSTRACT: Background: Inhibition of nitric oxide (NO) synthase by L-arginine analogs is associated with elevation of blood pressure in rats. Because endotheliumdependent vasomotion in different vascular beds is not homogenous, the aim of this study was to characterize and compare regional hemodynamic responses in carotid, femoral, and renal vascular beds after chronic NO inhibition in spontaneously hypertensive rats. The possible role of circulating endothelin and renin angiotensin systems in mediating the effects of chronic NO inhibition was also studied. Methods: Systemic and regional hemodynamics, left ventricular mass, plasma renin activity, and plasma endothelin-1 were determined in control and NW-nitro-Larginine methyl ester (L-NAME)-treated (10 mglkglday, 4 weeks) spontaneously hypertensive rats. Results: L-NAME treatment increased arterial pressure and total peripheral and regional vascular resistance and decreased cardiac output, stroke volume, and regional blood flow. An in-
E
ndothelium is the major source of the endothelium-derived relaxing factor nitric oxide (NO), which affects contractile function 1 and proliferation and migration of vascular smooth muscle cells. 2 NO is derived from L-arginine in a process that can be competitively inhibited by such L-arginine analogs as NW-nitro-L-arginine methyl ester (L-NAME),1 Inhibition of NO synthesis causes vasoconstriction,3
From the Institute for Medical Research (JV, MJ, DJ, JM-L, VL, Medicine-Clinical Center
GR-I(, DK) and the Institute of Nuclear (DN-M, GA-G), Belgrade, Yugoslavia.
Submitted November 11, 1999; accepted in revised form February 15, 2000. This work was supported by a grant from the Ministry of Science and Technology of Serbia. Correspondence: Jasmina Varagic M.D., Ph.D., Hypertension Research, Alton Ochsner Medical Foundation, 1520 Jefferson Highway, New Orleans, LA 70121. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
crease in blood flow ratio and a decrease in vascular resistance ratio between carotid and renal as well as femoral and renal vascular beds in rats treated with L-NAME was found. Blood flow and vascular resistance ratios between femoral and carotid vascular beds remained unchanged. L-NAME increased plasma renin activity and left ventricular weightlbody weight ratio, whereas plasma endothelin-1 was not modified. Conclusions: The results of this study showed that the renal circulation seemed to be more sensitive to the effects of chronic NO inhibition than carotid and femoral vascular beds. Simultaneous activation of the renin angiotensin system may further potentiate cardiovascular effects of chronic NO inhibition. No evidence that circulating endothelin-1 plays a role in this model of hypertension was found. KEY INDEXING TERMS: Nitric oxide; Regional hemodynamics; Plasma renin activity; Endothelin; Spontaneously hypertensive rats. [Am J Med Sci 2000;320(3):171-6.]
increase in total peripheral resistance, and impairment in regional blood flOWS. 4- 7 Previous studies have demonstrated that NO has an important role in long-term regulation of arterial blood pressure in spontaneously hypertensive rats (SHR).8,9 It has been shown that chronic administration of the L-arginine analog L-NAME in SHR induced a significant rise in blood pressure accompanied with decreased cardiac output and increased total peripheral resistance. The effects of chronic NO synthase inhibition on renal9,10 and coronary circulationl l have also been studied. However, the hemodynamic response of other vascular beds to the chronic administration of L-NAME has not been examined in SHR. Because endothelium-dependent vasomotion in different vascular beds is not homogenous,12-14 this study was designed to characterize and compare regional hemodynamic responses in carotid, femoral, and renal vascular beds after chronic NO synthesis inhibition in SHR. Further171
Nitric Oxide Blockade in Spontaneously Hypertensive Rats
more, the attempt was made to delineate the possible role of circulating endothelin and renin-angiotensin system in mediating the effects of chronic NO synthesis inhibition.
240
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Material and Methods The protocol of the present study was approved by the Institutional Committee on Animal Care and Use of Laboratory Animals of Institute for Medical Research, Belgrade, Yugoslavia. Experimental Groups. Experiments were performed in adult male SHR weighing about 250 g. The rats were divided into 2 experimental groups: 1 group was given L-NAME in drinking water (average dose, 10 mg/kg/day; fresh solutions prepared daily) for 4 weeks; rats in the control group were given tap water. There were 24 rats in each group. All animals were given standard food for laboratory rats (Veterinarski zavod, Zemun, Yugoslavia). Experimental Protocol. Two separate baseline determinations of body weight and systolic blood pressure (tail-cuID were made in a span of 2 weeks in all rats. Afterward, animals were assigned randomly to their respective treatments for a period of 4 weeks. Measurements of blood pressure were made once a week during the 4-week course of the treatment. At the end of the fourth week, hemodynamic studies were done in 16 rats selected randomly from each group. In the remaining rats from each group, measurements of plasma endothelin-1 (ET-1) and plasma renin activity (PRA) were performed. At the end of the experiment, all rats were exsanguinated and their hearts and left kidneys removed. The atria were excised, the lateral right ventricular wall was separated from the left ventricle and septum, and their weights were determined on an automatic analytical balance. The ratio ofthe left and right ventricular weights as well as of the left kidney weight to body weight was determined. Experimental Techniques. Indirect determinations of systolic blood pressure were made using a tail-cuff pneumatic pulse detector and a recorder (Narco Bio-System, Houston, TX). For hemodynamic studies, rats were anesthetized with pentobarbital (35 mg/kg, intraperitoneal injection) and a tracheal cannula was inserted. Blood pressure was measured directly through a femoral artery catheter PE-50 with a low-volume displacement transducer P23Db and a direct writing recorder (Physiograph Four; Narco Bio-System). Mean arterial pressure (MAP) was obtained by electronic integration. Cardiac output (CO) was determined by a previously described15 modification of Coleman's16 application of the dye dilution technique. Indocyanine green was used as the indicator, and a recording densitometer (Beckman Instruments, Columbia, MD) was used for the determination of dye in the blood and registration of the dilution curve. Total peripheral resistance (TPR) was calculated from the MAP and CO (assuming that the mean right atrial pressure is zero) and is expressed as millimeters of Hg per milliliter per minute per 100 g of body weight. Stroke volume was calculated by dividing CO by heart rate. For blood flow measurement, femoral, carotid and renal arteries were carefully isolated. An ultrasonic flow probe (lRB, internal dimeter, 1 mm) was placed around the arteries for the measurement of total femoral (FBF), carotid (CBF), and renal blood flow (RBF) using a Transonic T106 Small Animal Bloodflow meter (Transonic Systems, Ithaca, NY). Regional blood flow was normalized to flow in milliliter per minute per 100 g of body weight. Regional vascular resistance (VR) was calculated by dividing the MAP by the regional blood flow and is expressed as millimeters of Hg per milliliter per minute per 100 g of body weight. From the blood samples collected from the abdominal aorta, hematocrit (Hct), and plasma creatinine concentrations were determined using the micromethod and Jaffe reaction. For determinations of plasma ET-1 and PRA, the animals were decapitated and blood samples were collected into chilled, siliconized centrifuge tubes containing Na-EDTA (1 mg/mL) and aprotinin (1000 kIU/mL) for ET-1 and Na-EDTA (2 mg/mL) for PRA. RPA 5359 Endothelin-1,2 biotrack assay system (provided
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Results
L-NAME treatment had no influence on body weight. Mean body weight at the end of experiment was 290 ± 6 g in control rats and 277 ± 7 g in L-NAME group. Changes in the systolic blood pressure during the 4 weeks oftreatment are shown in Figure 1. Systolic blood pressure rose progressively in L-NAMEtreated rats, reaching 232 ± 3 mm Hg by the end of the treatment, compared with 179 ± 2 mm Hg in untreated control rats. Mter the 4-week treatment, L-NAME significantly increased MAP. This elevation of arterial pressure in rats drinking L-NAME was accompanied by a significantly higher TPR and a substantial reduction in CO (Figure 2). No change in heart rate (368 ± 4 in control rats versus 365 ± 15 bpm in L-NAME-treated SHR) and Het (48 ± 2 in control rats versus 49 ± 3% in L-NAME-treated SHR) was found, whereas stroke volume decreased significantly in SHR given L-NAME (0.070 ± 0.007 in control rats versus 0.047 ± 0.005 mUbeatl100 gin L-NAME-treated SHR; P < 0.05). In rats drinking L-NAME, blood flow was significantly reduced and VR was increased in all vascular beds studied compared with the control rats (Figures 3, 4, and 5). Blood flow and VR ratios between tissue regions within the control and L-NAME group are shown in Fig 6. Intergroup evaluation revealed an increase in blood flow ratio and a decrease in VR ratio between carotid and renal as well as femoral and renal vasSeptember 2000 Volume 320 Number 3
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Figure 5. Changes in renal vascular bed in response to chronic NO synthesis inhibition; ***, P < 0.001; n=16 for each group. RVR, renal vascular resistance.
cular beds in rats treated with L-NAME. On the other hand, blood flow and VR ratio between femoral and carotid vascular beds remained unchanged after prolonged NO synthesis inhibition. These results
showed that vasoconstriction was more pronounced in renal vasculature than in the other vascular beds studied. Plasma creatinine level was higher after 4-week treatment with L-NAME (78.90 ± 3.61 in
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173
Nitric Oxide Blockade in Spontaneously Hypertensive Rats
Table 1. Organ Weights in SHR Given L-NAME
LVW (mg/100 g) RVW (mg/100 g) LKW (mg/100 g)
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314.55 ± 4.29 67.44 ± 2.26 340.03 ± 7.03
331.38 ± 6.38* 62.32 ± 2.76 337.17 ± 18.40
L VW, left ventricular weight/body weight ratio; RVW, right ventricular weight/body weight ratio; LKW, left kidney weight/body weight ratio. Values are means ± SEM; n = 24 for each group; *p < 0.05.
control versus 112.22 ± 12.27 mmollL in L-NAMEtreated rats; P = 0.057), although the difference between groups was not statistically significant. Chronic oral administration of L-NAME induced increase in PRA (1.6 ± 0.2 in control rats versus 24.65 ± 2.2 ng of angiotensin 1/mI1hr in L-NAMEtreated rats, P < 0.001), whereas plasma ET-1 was not modified by L-NAME treatment (27.2 ± 2.4 in control rats versus 21.6 ± 3.6 fmollmL in L-NAMEtreated rats). Left ventricular weightlbody weight ratio was found to be significantly increased in L-NAMEtreated rats, whereas right ventricular weightlbody weight ratio and left kidney weightlbody weight ratio of rats given L-NAME were not different from those in control SHR (Table 1). However, no correlation between PRA and left ventricular weightlbody weight ratio was found (r = 0.306). Discussion
The present results show that after treatment with L-NAME, VR is increased and blood flow decreased in all vascular beds studied. Furthermore, vasoconstriction was more pronounced in renal vasculature than in 2 other vascular beds studied, as indicated by a decrease in carotid VRfrenal VR and femoral VRfrenal VR ratios and an increase in CBFI RBF and FBFIRBF ratios. Therefore, our results suggest that inhibition of NO-mediated mechanisms contribute differently to the vascular tone in different vascular beds. Several in vitro and in vivo studies have shown that L-NAME increased sensitivity of renal arterioles to the vasoconstrictor effect of angiotensin 11.17,18 It seems likely that the combination of decreased NO synthesis with activated reninangiotensin system made renal circulation the most sensitive among the 3 vascular beds studied. A higher plasma creatinine concentration revealed decreased glomerular filtration rate that was also reported in few previous studies. 9,10 Carotid and femoral circulation displayed similar sensitivity to chronic NO synthesis inhibition. Prolonged inhibition of NO synthesis resulted in a severe elevation of arterial pressure and was associated with an increase in total peripheral resistance 174
and a decrease in cardiac output, suggesting that NO has an important role in long term control of arterial pressure in SHR. These results are in agreement with earlier reports. 8,9,19 Moreno et al. suggested that impairment of CO might be a consequence of a decreased contractility caused by myocardial ischemia. An increased work demand and a decreased coronary flow may lead to ischemia and impaired contractility in this model ofhypertension.19 On the other hand, in an acute study, inhibition of NO synthesis reduced coronary flow but did not affect myocardial oxygen consumption. 20 It should also be mentioned that after prolonged inhibition of NO synthesis, no signs of heart failure (weakness, pulmonary edema, hepatomegaly, ascites) were reported. It is possible that vasoconstriction induced by chronic NO synthesis inhibition does not result solely from removal of basal NO release but also from activation of vasoconstrictors; it has been shown that NO donors reduce basal and stimulated ET production. 21 Therefore, it may be expected that inhibition of NO synthesis would stimulate ET production. However, our results show that plasma ET-1 levels were not affected significantly by LNAME, indicating that circulating ET-1 does not have a role in progressive rise of blood pressure and the development of left ventricular hypertrophy in this model. In the other 2 studies, carried out in SHR, 3 weeks' treatment with L-NAME induced a slight increase in circulating ET.22,23 The differences between our results and those of other studies may be attributable to the differences in doses of LNAME. Furthermore, it was demonstrated that blood pressure elevation in SHR treated with LNAME is endothelin independent, because no response to chronic endothelin receptor blockade was observed. 23 Thus, it does not seem likely that ET plays a role in L-NAME-induced hypertension. The present results also showed that chronic NO synthesis inhibition in SHR increased PRA, which may further aggravate hypertension and ventricular hypertrophy. The data on the effect of NO synthesis inhibition on PRA are contradictory24-28; it may be suggested that renin secretion increases only after prolonged blockade of NO synthesis, which is associated with development of severe hypertension and vascular lesions in the kidneys. This has been previously described in normotensive rats given high doses of L-NAME28 and in SHR treated with 20 mg/kg/day of L-NAME.29 In the present study, a significant increase in left ventricular mass was also found in rats given LNAME. The reports concerning the development of left ventricular hypertrophy in experimental model of chronic NO synthesis inhibition8- 11 ,22,23,29,30 are conflicting. A number of studies show that in rats treated with inhibitors of NO synthesis, a significant increase in arterial pressure occurs, but left ventricSeptember 2000 Volume 320 Number 3
Vorogic at 01
ular hypertrophy develops only in animals with increased PRA.8,28-30 In contrast, no increase in left ventricular mass in L-NAME-treated SHR despite increased PRA has also been reported. 22 ,23 In our study, PRA was increased (range, 7.25-36 ng of angiotensin l/mUhr), but intragroup evaluation did not reveal any correlation between PRA and left ventricular mass in the L-NAME group. It is possible that 3 weeks ofL-NAME treatment was not long enough to produce a more prominent change in left ventricular mass, as noted by Moreno et al. 19 They found that the increase in left ventricular weight index in the L-NAME group was significant after 8 weeks but not in rats studied after 2 or 4 weeks of treatment. Indeed, only 5% increase in left ventricular mass in L-NAME group was found in our study. On the other hand, intense myocardial alteration (myocardial necrosis and fibrosis), frequently reported early in the course ofL-NAME treatment,19,31 may also influence left ventricular mass. The extent and duration of NO synthesis inhibition may be one of the factors that determined the development of left ventricular hypertrophy in this experimental model. All these factors complicated the interpretation of the data, but our results of significant differences in left ventricular weightlbody weight ratio and PRA between the control and L-NAME groups seem to support the thesis that the renin angiotensin system plays an important role in the pathogenesis of the cardiac hypertrophy in this experimental model. In summary, the results of this study showed that NO has an important role in the long-term regulation of arterial blood pressure and regional blood flow. It seems likely that simultaneous activation of the renin angiotensin system further potentiated cardiovascular effects of chronic NO inhibition. Compared with carotid and femoral vascular beds, renal circulation seemed to be more sensitive to the effects of chronic NO inhibition.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16. 17.
Acknowledgements:
18.
The expert technical assistance of Mrs. Zaga Jovanovic is acknowledged. We thank Dr. Dinko Susic for helpful comments and suggestions.
19.
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28. Zanchi A, Schaad N, Osterheld M-C, et at. Effects of chronic NO synthase inhibition in rats on renin-angiotensin system and sympathetic nervous system. Am J Physiol 1995; 268:H2267-73. 29. Vaskonen T, Mervaala E, Krogerus L, et al. Cardiovascular effects of chronic inhibition of nitric oxide synthesis and dietary salt in spontaneously hypertensive rats. Hypertens Res 1997;20:183-92. 30. Arnal JF, EI Amrani AI, Chatellier G, et at. Cardiac weight in hypertension induced by nitric oxide synthase blockade. Hypertension 1993;22:380-7. 31. Numaguchi K, Egashira K, Takemoto M, et al. Chronic inhibition of nitric oxide synthesis causes coronary microvascular remodeling in rats. Hypertension 1995;26:957-62.
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