The long-term blockade of nitric oxide synthase (NOS) produces not only hypertension but also pathological structural changes of the coronary vasculature and fibrosis of the myocardium in animals. (1,2). Impaired endothelium-dependent relaxation has been shown also in experimental and human hypertension (3,4), and this phenomenon may be due in great part to decreased release or activity of nitric oxide (NO) (5). We have reported that some antihypertensive drugs may improve the impaired NOS activity in hypertensive rats (6,7). Recent studies (8,9) have reported that angiotensin II type 1 (AT1)-receptor antagonist attenuates the development of left ventricular hypertrophy and hypertrophic cardiac structural changes. However, it is not known whether NO is involved in beneficial effects of AT1-receptor antagonist. The purpose of this study was to evaluate the effects of long-term treatment with TCV-116, an AT1-receptor antagonist, on endothelial cell (e) NOS mRNA and protein expression and NOS activity in the left ventricle (LV), and its relation to myocardial remodeling including type I collagen mRNA expression in two-kidney, one-clip Goldblatt hypertensive rats.
Animal models and experimental designs
Twenty-two male Sprague-Dawley rats (Oriental Bioservice Kanto Inc., Ibaragi, Japan), aged 40-45 days, were used in this study. Two-kidney, one-clip Goldblatt hypertension (RHR) was induced in 15 rats by placing a silver clip (0.20 mm internal width) on the left renal artery while the rats were under light ether anesthesia, leaving the right kidney untouched (6,10). Systolic blood pressure (SBP) was measured by the tail-cuff method (model MK-1100; Muromachi Kikai, Tokyo, Japan) before the operation and at 1-week intervals thereafter. Four weeks after clipping. RHR were randomly assigned to either the TCV-116 (3 mg/kg/day, subdepressor dose, Takeda Chemical Industries, Ltd, Osaka, Japan) administered p.o. in a volume of 2 ml/kg as a gum arabic suspension (RHR-TCV: n = 8) or the vehicle given (RHR-V: n = 7) for 6 weeks. Age-matched rats subjected to a sham operation also were administered the vehicle for 6 weeks (ShC, n = 7) (protocol 1). Numbers of animal model and experimental designs of protocols 2, 3, and 4 were treated in the same manner as described for protocol 1. Rats were housed at a constant temperature (25 ± 1°C), and fed a standard laboratory rat chow (0.4% sodium content).
Reverse transcription-polymerase chain reaction. After 6 weeks of treatment, the rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and decapitated, and the heart was immediately excised. The LV was carefully separated from the atria and right ventricle, weighed, immediately frozen in liquid nitrogen, and stored at −80°C until extraction of total RNA. Total RNA was prepared as previously described (11). Reverse transcription-polymerase chain reaction (RT-PCR) was performed by standard methods with 1 μg of total RNA (12). First-strand cDNA was synthesized with random primers and Molony murine leukemia virus reverse transcriptase (Promega, Madison, WI, U.S.A.), and PCR amplification was then performed with synthetic gene-specific primers for eNOS (upstream primer, 5′-TCCAGTAACACAGACAGTGCA-3′; downstream primer, 5′-CAGGAAGTAAGTGAGAGC-3′; product length, 693 bp) (13) and type I collagen (upstream primer, 5′-TGTTCGTGGTTCTCAGGGTAG-3′; downstream primer, 5′-TTGTCGTAGCAGGGTTCTTTC-3′; product length, 254 bp) (14) by using a DNA PCR kit (Perkin Elmer, Norwalk, CT, U.S.A.) for 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C for 1 min. Parallel amplification of rat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was performed for reference with primers as described (15). Reaction conditions were optimized to obtain reproducible and reliable amplification within the logarithmic phase of the reaction, as determined by preliminary experiments. The reaction was linear to 35 cycles with use of the ethidium bromide detection method. PCR products were separated by electrophoresis on a 2% agarose gel containing ethidium bromide and were visualized by ultraviolet-induced fluorescence. The intensity of each band was quantified by using a densitometer. The eNOS and type I collagen signals were normalized relative to the corresponding GAPDH signal from the same RNA sample (6,7,16,17).
Histologic examination and evaluation of myocardial remodeling. Histologic examination was studied as described in detail previously (6,7,10,17). In brief, excised hearts were perfused with physiologic saline solution containing adenosine, 10 μg/kg, and nitroglycerin, 10 μg/kg, and then with 6% formaldehyde solution via retrograde infusion into the ascending aorta at a pressure of 90 mm Hg. The LV was separated from the right ventricle, the atria, and the great vessels, and cut into five pieces perpendicular to the long axis. For light microscopy, 1.5-μm-thick sections were cut (Microtome, Tokyo, Japan). Paraffin slices from each heart were mounted on glass slides and stained with hematoxylin-eosin and Masson's trichrome stains. All histopathologic sections of each animal were examined by using a 3CCD color video camera (model DXC-930; Sony, Tokyo, Japan) mounted on a standard microscope (BHS-F; Olympus, Tokyo, Japan). Drawings of the limits of the vessels were made on the screen of a multiscan color computer display (model CPD-17SF7; Sony) and then digitized with a two-dimensional analysis system (Mac SCOPE; Mitani Corporation, Fukui, Japan) connected with a Macintosh computer system (Power Macintosh G3; Apple Computer Inc., Cupertino, CA, U.S.A.). Histopathologic findings of the myocardium and coronary arterioles were examined. We always measured the capillary density and cross-sectional surface area in the endocardium of the posterior portion of the left ventricular free wall. In this part of the heart, shrinkage was minimal, and orientation of the myocardial fibers was similar from one heart to another. We analyzed five sites from each ventricle in all rats. To assess thickening of the coronary arterial wall and perivascular fibrosis, the transsectional images of the area of the total small arteriolar lumen ≤104 μm2 were studied. The inner border of the lumen and the outer border of the tunica media were traced in each arterial image with hematoxylin-eosin staining at ×100 to ×400 magnification, and the areas encircled by the tracings were calculated. In quantification, non-round vessels resulting from oblique transsection or branching were excluded, and only round vessels were studied. The wall-to-lumen ratio (the area of the vessel wall divided by the area of the total blood vessel lumen) was determined. The area of fibrosis immediately surrounding blood vessels was calculated, and perivascular fibrosis was determined as the ratio of the area of fibrosis surrounding the vessel wall to the total area of the vessel. To assess the area of myocardial fibrosis, the area of pathological collagen deposition was measured in the microscopic field of each Masson's trichrome-stained section. The ratio of the total area of fibrosis within the left ventricular myocardium to the total area of the left ventricular myocardium in each heart was calculated and was used for analysis. The histopathologic examination on the sections from each rat was carried out by an operator who was blinded to the treatment groups.
Nitrite production in myocardium slices. On the day of the experiment, the rats were anesthetized with an intraperitoneal injection of pentobarbital (30 mg/kg body wt.). The right carotid artery was cannulated, and a PE-50 tube was inserted into the LV. The heart was perfused with physiologic saline and then with periodate-lysine-paraformaldehyde solution. The heart was taken out immediately, and fat and fibrous tissue, the great vessel, atria, and right ventricular free wall were removed in this order. After being rinsed with periodate-lysine-paraformaldehyde, the tissue was used for the assay of nitrite production within 24 h. Three 50-μm sections of each myocardium were cut on a vibratome and incubated in a buffer (pH 7.2) containing 25 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES; Sigma, St. Louis, MO, U.S.A.), 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM glucose for 48 h at 37°C. The supernatant was used for the assay of NO2− production, and the amount of NO2− was corrected by protein amount measured by the Bradford method (Bio-Rad, Richmond, CA, U.S.A.). Nitrite was measured by an autoanalyzer (TCI-NOX 1000 m; Tokyo Kasei Kogyo, Tokyo, Japan) by using the Griess method (6,17-19).
Western blot analysis. LV was homogenized (25% wt/vol) in 10 mM HEPES buffer, pH 7.4, containing 320 mM sucrose, 1 mM EDTA, 1 mM DTT, 10 μg/ml leupeptin, and 2 μg/ml aprotinin at 0-4°C with a polytron homogenizer. Homogenate was centrifuged at 1,000 g for 5 min at 4°C, and the resulting supernatant was used as a postnuclear fraction. Protein concentrations were determined with bovine serum albumin as a standard protein (20).
The postnuclear fraction (eNOS: 50 μg of protein) of sample was subjected to SDS-PAGE with 10% gels (21). The proteins in the gels were transferred electrophoretically to PVDF sheets for 1 h at 2 mA/cm2 as described (22). The sheets were immunoblotted with an anti-eNOS antibody (Transduction Laboratories) in a buffer containing 10 mM Tris/HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20, and 5% skim milk followed by peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG; Amersham Life Science Inc.) (22). The eNOS proteins transferred to the sheets were detected by using the ECL immunoblotting detection system (Amersham Life Science Inc). The amount of protein was quantified by using a densitometer in a linear range and expressed as percentage relative to that in nontreated rat.
All results are expressed as mean ± SEM. The mean values were compared among the three groups by using analysis of variance (ANOVA) followed by the Bonferroni test. Differences of p < 0.05 were considered statistically significant. Calculations, including those of derived values, and statistical tests, were performed by using the appropriate software (Stat View-J 4.5; Abacus Concepts Inc., Berkeley, CA, U.S.A.) and a Power Macintosh computer system (G3; Apple Computer Inc., Cupertino, CA, U.S.A.).
Systemic hemodynamics, body weight, and left ventricular weight
Before treatment with TCV-116 (4 weeks after clipping), SBP, measured by the tail-cuff method, was 128 ± 3 mm Hg in ShC, 205 ± 6 mm Hg in RHR-V, and 203 ± 5 mm Hg in RHR-TCV. As shown in Table 1, SBP in RHR-V and RHR-TCV after 6 weeks of treatment was similar and significantly higher than that in ShC. Heart rate was similar in ShC and RHR-V, and was not changed by the administration of the TCV-116. Body weight also was similar among the three groups. The LV mass of the RHR-V was significantly increased compared with that of ShC in body weight-corrected values. Long-term treatment with TCV-116 did not completely normalize the LV mass in RHR-TCV, but significantly decreased in RHR-TCV (25.7%) compared with the RHR-V after 6 weeks of treatment with TCV-116.
RT-PCR for left ventricular eNOS, type I collagen mRNA expression
The level of eNOS mRNA in the LV was significantly decreased in RHR-V compared with ShC (0.14 ± 0.02 vs. 0.31 ± 0.04, eNOS mRNA/GAPDH mRNA, p < 0.01), and significantly increased in RHR-TCV compared with ShC and RHR-V (0.51 ± 0.06 vs. ShC and RHR-V, p < 0.01, respectively; Figs. 1 and 2A). Type I collagen mRNA was significantly greater in RHR-V than in ShC (0.71 ± 0.07 vs. 0.38 ± 0.03, type I collagen mRNA/GAPDH mRNA, p < 0.01), and was significantly less in RHR-TCV than in RHR-V (0.49 ± 0.04 vs. RHR-V, p < 0.01; Figs. 1 and 2B).
Microscopic pictures taken of small coronary arteries with Masson's trichrome stain for the ShC, RHR-V, and RHR-TCV groups are shown in Fig. 3. The wall-to-lumen ratio increased in RHR-V compared with ShC and was significantly decreased by TCV-116 treatment (Fig. 4A). The degree of perivascular fibrosis was significantly greater in RHR-V than in ShC, and also was significantly decreased by TCV-116 treatment (Fig. 4B). Compared with ShC, myocardial fibrosis was significantly greater in RHR-V, and significantly less in RHR-TCV than in RHR-V (Fig. 4C).
Nitrite production in the myocardium slices
The myocardium slices of RHR-V produced significantly less NO2− than those in ShC (2.44 ± 0.29 vs. 3.81 ± 0.26 nmol/mg protein; p < 0.01). The NO2− production was significantly increased in RHR-TCV compared with RHR-V and ShC (5.87 ± 0.43 nmol/mg protein vs. ShC and RHR-V; p < 0.01, respectively; Fig. 5).
Western blot analysis
The eNOS protein mass in the LV was significantly decreased in RHR-V compared with ShC (368.1 ± 35.9 vs. 619.3 ± 57.4; p < 0.05), and significantly increased in RHR-TCV compared with RHR-V (914.8 ± 86.3 vs. RHR-V; p < 0.05; Fig. 6).
This study has indicated that downregulation of eNOS mRNA and protein expression and NOS activity in the LV may contribute to the development of the myocardial structural changes, and that production of eNOS mRNA and protein expression, NOS activity, and suppression of type I collagen mRNA expression by TCV-116 for 6 weeks may have a key role in the beneficial effects of cardioprotective action, because the blood pressure in RHR-V and RHR-TCV is at similar levels. Several studies have reported that effective antihypertensive therapies have restored the impaired endothelial function (23,24). We have reported that some antihypertensive drugs may improve the impaired NOS activity in hypertensive rats (6,7,17-19). However, effective antihypertensive therapies have not always improved the impaired endothelium-dependent relaxation (25), suggesting a difference in the ability of antihypertensive drugs to restore NOS activity. In our study, we have shown that AT1-receptor antagonist stimulates the expression of eNOS mRNA and protein. There are possible mechanisms by which AT1-receptor antagonist has an increased NO production. One possible explanation is that Ang II produces vascular and myocardial fibroproliferative changes as well as to inhibit NOS directly via an action on the AT1-receptor subtype (26), thus the AT1-receptor antagonist increases the NOS expression (27-29). The local renin-angiotensin system in the LV may be involved in pathological cardiac hypertrophy and remodeling through the AT1 receptor in vivo, and AT1-receptor antagonists potently inhibit the expression of the growth-related and extracellular matrix genes, as well as cellular phenotypic modulation. Taken together, these effects of the AT1-receptor antagonist may contribute to an increased NO production and protective effects on impairment of coronary circulation (28-30). Another possible explanation is that AT1-receptor antagonists have been demonstrated to induce a dose-dependent enhancement of plasma Ang II levels due to inhibition of the negative-feedback regulation of renin release from the juxtaglomerular apparatus in the kidney (31). The accumulated Ang II may excessively stimulate Ang II type 2 receptor (32). Therefore, AT1-receptor antagonists block the AT1 receptor-mediated effects of Ang II but induce Ang II accumulation and, possibly, a stimulation of Ang II type 2 receptors, leading to an increased NOS production (9,33,34).
We speculate that blockade of the AT1 receptor increased the Ang II concentration (35), which may then activate the AT2 receptor (36,37). AT2-receptor activation, in turn, increased NO release (38). These effects of AT1-receptor blockade were abolished by blockade of the bradykinin B2 receptor (37,38). Moreover, the beneficial effects of AT1-receptor antagonist on ventricular remodeling were abolished by the AT2-receptor blocker PD123319 and, similar to the effects of the angiotensin-converting enzyme inhibitor in the same study, by the bradykinin B2 receptor blocker HOE 140 (36). Thus the activation of the AT2 receptor was obviously crucial for the beneficial effects achieved by AT1-receptor blockade. However, further studies are needed to elucidate the mechanism by which upregulation of eNOS expression may be due in part to an increased AT2-receptor stimulation.
To evaluate whether TCV-116 is a special AT1-receptor antagonist to have a potency to increase NO production by a subdepressor dose of TCV-116, we performed the supplemented experiments of protocol 1 in six age-matched male Sprague-Dawley rats subjected to a sham operation, with the TCV-116 administered p.o. in a volume of 2 ml/kg as a gum arabic suspension at a dose of 3 mg/kg/day for 6 weeks (ShC-TCV, n = 6). The level of eNOS mRNA expression in the LV was significantly increased in ShC-TCV compared with ShC (0.64 ± 0.05 vs. 0.31 ± 0.04, eNOS mRNA/GAPDH mRNA; p < 0.01). These results indicate that the enhancement of the effects of NO may be related to an increase in eNOS mRNA expression in age-matched Sprague-Dawley rats treated with subdepressor dose of TCV-116, and that TCV-116 may have a potency to increase NO production independent of blood pressure.
It has been proposed that various forms of hypertension are characterized by a dysfunctional endothelium. It is indicated that a deficient production of endothelium-derived NO results in diminished vasodilator tone, allowing vascular resistance to increase, and this contributes to the elevated blood pressure (39,40). It has been suggested that sustained hypertension results in endothelial damage or dysfunction and that the resulting decrease in NO exacerbates the underlying hypertension (41,42). Several experimental models of hypertension have demonstrated that endothelium-dependent vasodilation is impaired. Chou et al. (43) demonstrated that an early decline (from 4 to 14 weeks) in eNOS activity and protein expression in the aorta was similar in both spontaneously hypertensive (SHR) and Wister-Kyoto (WKY) rats, and that in the aging process (from 14 to 63 weeks), the eNOS activity and protein expression of SHR was significantly lower than that of WKY rats. Stroke-prone SHR (SHRSP), in which hypertensive organ damage is more marked than in SHR, have been regarded as an experimental model for human malignant hypertension (44). Endothelium-dependent vasodilation also is depressed in this strain of rats (5,45). In addition, Malinski et al. (46) examined bradykinin-induced NO release from cultured endothelial cells of SHRSP and found it decreased compared with that from cells of WKY rats. On the other hand, Hoshino et al. (47) suggested that their findings were enhanced vasoconstriction to norepinephrine and depressed endothelium-dependent relaxations to acetylcholine in the aortic rings from one-kidney, one-clip renovascular hypertensive rats compared with control rats. We have recently shown that NOS activity and eNOS mRNA expression in the LV were significantly decreased in two-kidney, one-clip Goldblatt hypertensive rats compared with sham-operated control (6). Furthermore, we have shown that NOS activity in rat kidney (18) and eNOS mRNA expression in the LV (17) were significantly decreased in long-term NO blockade-induced hypertensive rats. Moreover, we have reported that NOS activity in rat kidney was significantly decreased in deoxycorticosterone acetate-salt hypertensive rats (19). These findings suggest that the endothelial dysfunction may contribute to the hypertension in several experimental models because of a reduced production of NO.
This study demonstrated that RHR-V showed a significant increase of the wall-to-lumen ratio, perivascular fibrosis, myocardial fibrosis and fibrosis factor, type I collagen mRNA expression, with downregulation of eNOS mRNA and protein expression and NOS activity. Takemoto et al. (8,48) suggested that long-term blockade of NO synthesis by the prolonged administration of Nω-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthesis, produced systemic arterial hypertension, vascular structural changes, and fibrosis of the LV in rats. Numaguchi et al. (2) showed that prolonged inhibition of NO synthesis with L-NAME caused coronary microvascular remodeling and cardiac hypertrophy in rats in vivo. Furthermore, long-term intracoronary infusion of L-NAME, which had no effect on systemic arterial pressure, caused microvascular structural changes in pigs (49). Moreover, recently we also demonstrated that long-term blockade of NO synthesis produced coronary microvascular remodeling, upregulation of type I collagen mRNA expression, and downregulation of eNOS mRNA expression and NOS activity in L-NAME-induced hypertensive rats (17). These results indicate that these decrease eNOS mRNA and protein expression and NOS activity may play a role in the progression of coronary microvascular remodeling in RHR. In addition, a subdepressor dose of TCV-116 improved these myocardial remodelings in RHR, which may be due in part to an increased eNOS mRNA and protein expression and NOS activity in the LV.
In conclusion, we evaluated the effects of long-term treatment with TCV-116 on eNOS mRNA and protein expression, NOS activity in the LV, and its relation to myocardial remodeling including type I collagen gene expression in two-kidney, one-clip Goldblatt hypertensive rats. These results showed that downregulation of eNOS mRNA and protein expression, and NOS activity in RHR-V was significantly reversed in the LV by TCV-116 treatment. Myocardial remodeling of Goldblatt hypertensive rats was significantly ameliorated by a subdepressor dose of TCV-116, which may be due to an increased eNOS mRNA and protein expression and NOS activity in the LV.
Acknowledgment: This work was supported by a grant from the Ueda Memorial Trust Fund for Research of Heart Diseases, Tokyo, Japan. We thank Kazumi Akimoto for technical assistance with RT-PCR, Mrs. Sachie Sato for technical assistance with Western blotting, Mrs. Noriko Suzuki for preparing and staining tissue sections for histologic investigation, and Miss Yasuko Mamada for technical assistance. We thank Takeda Chemical Industries for supplying TCV-116.
1. Ribeiro MO, Antunes E, Nicci G, Lovisolo SM, Zatz R. Chronic inhibition of nitric oxide synthesis: a new model of arterial hypertension. Hypertension
2. Numaguchi K, Egashira K, Takemoto M, et al. Chronic inhibition of nitric oxide synthesis causes coronary microvascular remodeling
in rats. Hypertension
3. Konishi M, Su C. Role of endothelium in dilator responses of spontaneously hypertensive rat arteries. Hypertension
4. Panza JA, Casino PR, Kilcoyne RN, Quyyumi AA. Role of endothelium-derived nitric oxide in the abnormal endothelium-dependent vascular relaxation of patients with essential hypertension. Circulation
5. Hirata Y, Hayakawa H, Kakoki M, et al. Nitric oxide release from kidneys of hypertensive rats treated with imidapril. Hypertension
6. Kobayashi N, Kobayashi K, Hara K, et al. Benidipine stimulates nitric oxide synthase and improves coronary circulation in hypertensive rats. Am J Hypertens
7. Kobayashi N, Hara K, Watanabe S, et al. Effect of imidapril on myocardial remodeling
in L-NAME-induced hypertensive rats is associated with gene expression of NOS and ACE mRNA. Am J Hypertens
8. Takemoto M, Egashira K, Tomita H, et al. Chronic angiotensin-converting enzyme inhibition and angiotensin II type I receptor blockade: effects on cardiovascular remodeling
in rats induced by the long-term blockade of nitric oxide synthesis. Hypertension
9. Zhu YC, Zhu YZ, Gohlke P, Stauss HM, Unger T. Effects of angiotensin-converting enzyme inhibition and angiotensin II AT1
receptor antagonism on cardiac parameters in left ventricular hypertrophy. Am J Cardiol
10. Kobayashi N, Kobayashi K, Kouno K, Yagi S, Matsuoka H. Effect of benidipine on microvascular remodeling
and coronary flow reserve in two-kidney one clip Goldblatt hypertension. J Hypertens
11. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem
12. Hattori Y, Gross SS. GTP cyclohydrolase I mRNA is induced by LPS in vascular smooth muscle: characterization, sequence and relationship to nitric oxide synthase. Biochem Biophys Res Commun
13. Seki T, Naruse M, Naruse K, et al. Gene expression of endothelial type isoform of nitric oxide synthase in various tissues of stroke-prone spontaneously hypertensive rats. Hypertens Res
14. Nicoletti A, Heudes D, Hinglais N, et al. Left ventricular fibrosis in renovascular hypertensive rats: effect of losartan and spironolactone. Hypertension
15. Terada Y, Tomita K, Nonoguchi H, Marumo F. Polymerase chain reaction localization of constitutive nitric oxide synthase and soluble guanylate cyclase messenger RNAs in microdissected rat nephron segments. J Clin Invest
16. Hattori Y, Akimoto K, Murakami Y, Kasai K. Pyrrolidine dithiocarbamate inhibits cytokine-induced VCAM-1 gene expression in rat cardiac myocytes. Mol Cell Biochem
17. Kobayashi N, Yanaka H, Tojo A, Kobayashi K, Matsuoka H. Effects of amlodipine on nitric oxide synthase mRNA expression and coronary microcirculation in prolonged nitric oxide blockade-induced hypertensive rats. J Cardiovasc Pharmacol
18. Tojo A, Kobayashi N, Kimura K, et al. Effects of antihypertensive drugs on nitric oxide synthase activity in rat kidney. Kidney Int
19. Takanohashi A, Tojo A, Kobayashi N, Yagi S, Matsuoka H. Effect of trichlormethiazide and captopril on nitric oxide synthase activity in the kidney of deoxycorticosterone acetate-salt hypertensive rats. Jpn Heart J
20. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem
21. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature
22. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A
23. Tschudi MR, Criscione L, Novosel D, Pfeiffer K, Lüscher TF. Antihypertensive therapy augments endothelium-dependent relaxations in coronary arteries of spontaneously hypertensive rats. Circulation
24. Clozel M, Kuhn H, Hefti F. Effects of angiotensin converting enzyme inhibitors and of hydralazine on endothelial function in hypertensive rats. Hypertension
25. Panza JA, Quyyumi AA, Callahan TS, Epstein SE. Effect of antihypertensive treatment on endothelium-dependent vascular relaxation in patients with essential hypertension. J Am Coll Cardiol
26. Sadoshima J, Izumo S. Molecular characterization of angiotensin II-induced hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts: critical role of the AT1
receptor subtype. Circ Res
27. Tojo A, Madsen KM, Wilcox CS. Expression of immunoreactive nitric oxide synthase isoforms in rat kidney: effects of dietary salt and losartan. Jpn Heart J
28. Anderson IK, Drew GM. Investigation of the inhibitory effect of N
-(G)-nitro-L-arginine methyl ester on the antihypertensive effect of the angiotensin AT1
receptor antagonist, GR138950. Br J Pharmacol
29. Fujita H, Takeda K, Nakamura K, et al. Role of nitric oxide in impaired coronary circulation and improvement by angiotensin II receptor antagonist in spontaneously hypertensive rats. Clin Exp Pharmacol Physiol
30. Kim S, Iwao H. Involvement of angiotensin II in cardiovascular and renal injury: effects of an AT1
-receptor antagonist on gene expression and the cellular phenotype. J Hypertens
31. Campbell DJ, Kladis A, Valentijn AJ. Effects of losartan on angiotensin and bradykinin peptides and angiotensin-converting enzyme. J Cardiovasc Pharmacol
32. Unger T, Chung O, Csikos T, et al. Angiotensin receptors. J Hypertens
33. Siragy HM, Carey RM. The subtype 2 (AT2
) angiotensin receptor mediates renal production of nitric oxide in conscious rats. J Clin Invest
34. Maeso R, Navarro-Cid J, Muñoz-García R, et al. Losartan reduces phenylephrine constrictor response in aortic rings from spontaneously hypertensive rats: role of nitric oxide and angiotensin II type 2 receptors. Hypertension
35. Hubner R, Hogemann AM, Sunzel M, Riddell JG. Pharmacokinetics of candesartan after single and repeated doses of candesartan cilexetil in young and elderly healthy volunteers. J Hum Hypertens
36. Liu Y-H, Yang X-P, Sharov VG, et al. Effects of angiotensin-converting enzyme inhibitors and angiotensin II type 1 receptor antagonists in rats with heart failure: role of kinins and angiotensin II type 2 receptors. J Clin Invest
37. Wiemer G, Scholkens BA, Busse R, et al. The functional role of angiotensin II-subtype AT2
-receptors in endothelial cells and isolated ischemic rat hearts. Pharm Pharmacol Lett
38. Seyedi N, Xu XB, Nasjletti A, Hintze TH. Coronary kinin generation mediates nitric oxide release after angiotensin receptor stimulation. Hypertension
39. Lüscher TF, Vanhoutte PM. Endothelium dependent contraction to acetylcholine in the aorta of the spontaneously hypertensive rats. Hypertension
40. Lüscher TF, Raij L, Vanhoutte PM. Endothelium-dependent responses in normotensive and hypertensive Dahl rats. Hypertension
41. Dominiczak AF, Bohr DF. Nitric oxide and its putative role in hypertension. Hypertension
42. Peach MJ, Loeb AL. Changes in vascular endothelium and its function in systemic arterial hypertension. Am J Cardiol
43. Chou TC, Yen MH, Li CY, Ding YA. Alteration of nitric oxide synthase expression with aging and hypertension in rats. Hypertension
44. Yamori Y, Horie R, Sato M, Handa H. Pathogenetic similarity of stroke-prone SHR and humans. Stroke
45. Tesfamariam B, Halpern W. Endothelium-dependent and endothelium-independent vasodilation in resistance arteries form hypertensive rats. Hypertension
46. Malinski T, Kapturczak M, Dayharsh J, Bohr D. Nitric oxide synthase activity in genetic hypertension. Biochem Biophys Res Commun
47. Hoshino J, Sakamaki T, Nakamura T, et al. Exaggerated vascular response due to endothelial dysfunction and role of the renin-angiotensin system at early stage of renal hypertension in rats. Circ Res
48. Takemoto M, Egashira K, Usui M, et al. Important role of tissue angiotensin-converting enzyme activity in the pathogenesis of coronary vascular and myocardial structural changes induced by long-term blockade of nitric oxide synthesis in rats. J Clin Invest
49. Ito A, Egashira K, Kadokami T, et al. Chronic inhibition of endothelium-derived nitric oxide synthesis causes coronary microvascular structural changes and hyperreactivity to serotonin in pigs. Circulation