Cardiac and Vascular Effects of Long-term Losartan Treatment in Stroke-Prone Spontaneously Hypertensive Rats
In previous studies in stroke-prone spontaneously hypertensive rats (SHRSP), we demonstrated that early-onset, long-term angiotensin-converting enzyme inhibitor treatment improved cardiac function and metabolism and increased aortic cGMP content even at sub-antihypertensive doses. These effects could be prevented by bradykinin type 2 (B2) receptor blockade with icatibant. In the present study, we studied the effects of long-term oral treatment with the angiotensin type 1 (AT1) receptor antagonist losartan (30 mg/kg per day) on functional and biochemical parameters of the heart and on cGMP content in the aorta in SHRSP treated prenatally and subsequently up to the age of 20 weeks. Losartan prevented the development of hypertension and left ventricular hypertrophy. Cardiac function measured ex vivo in isolated perfused hearts was improved, as demonstrated by significant increases in left ventricular pressure (22.4%), differentiated left ventricular pressure (dP/dtmax) (35.1%), and coronary flow (38%). The release of the intracellular enzymes lactate dehydrogenase and creatine kinase and of lactate into the coronary effluent was reduced by 46.4%, 47.2%, and 63.6%, respectively. In myocardial tissue, the concentrations of glycogen and the energy-rich phosphates ATP and creatine phosphate were increased by 43.2%, 33.1%, and 42.4%, respectively, whereas lactate was decreased by 57.0%. The aortic tissue content of cGMP was increased fivefold. Our results demonstrate that chronic blockade of AT1 receptors with losartan improved cardiac function and metabolism and increased aortic cGMP content in SHRSP to an extent similar to that observed previously after long-term angiotensin-converting enzyme inhibitor treatment at a comparably antihypertensive dose. Prevention of hypertension and cardiac hypertrophy as well as stimulation of non-AT1 receptors are discussed to explain the cardiac and vascular actions of losartan.
- angiotensin-converting enzyme inhibitors
- rat, stroke-prone SHR
- receptors, angiotensin
- cyclic GMP
The peptide Ang II plays an important role in hypertension as well as cardiac and vascular disorders, and the inhibition of Ang II generation by ACE inhibitors has become an established approach to the treatment of these diseases. However, not all of the effects of ACE inhibition can be explained by the decrease in Ang II generation. Some of them, especially with respect to organ protection, have also been attributed to bradykinin potentiation.1 2
Recently, specific Ang II receptor antagonists have been developed to inhibit the renin-angiotensin system more specifically than ACE inhibitors. Two major subtypes of the Ang II receptor, AT1 and AT2, have been characterized, and some other subtypes have been described.3 Losartan, a specific antagonist of the AT1 receptor, is now clinically available for the treatment of hypertension. The antihypertensive action of losartan is based on the blockade of AT1 receptors, which are believed to mediate most of the cardiovascular actions of Ang II.4 A tissue-protective action of AT1 receptor blockade has been previously shown in studies in salt-loaded SHRSP, an animal model of malignant hypertension. In these rats, chronic AT1 blockade with losartan prevented stroke, malignant nephrosclerosis, and cardiac infarction and increased survival,5 6 7 effects similar to those observed after ACE inhibition8
In previous studies in SHRSP, we demonstrated beneficial actions of long-term ACE inhibition on cardiac function and metabolism in isolated perfused hearts.9 10 In these studies, early-onset, long-term treatment of SHRSP with the ACE inhibitors ramipril and perindopril increased cardiac contractility and coronary flow, reduced the release of the intracellular enzymes LDH and creatine kinase, and increased myocardial tissue levels of glycogen and the energy-rich phosphates ATP and creatine phosphate. In addition, the ACE inhibitors improved aortic function and increased NO production by the aortic endothelium, as evidenced by an increase in the aortic cGMP content.11 These effects of the ACE inhibitors were abolished after cotreatment with a bradykinin type 2 (B2) receptor antagonist, indicating that these cardiac and vascular actions of the ACE inhibitors depended on the inhibition of bradykinin breakdown rather than the reduction of Ang II generation. Our previous studies further suggested that the effects of the ACE inhibitors on cardiac and vascular functions were independent of their antihypertensive and antihypertrophic actions because they were also observed after sub-antihypertensive doses of the ACE inhibitors.
In the present study in SHRSP, we used losartan to selectively block the renin-angiotensin system at the level of the AT1 receptor without concomitant bradykinin potentiation. We show that long-term antihypertensive treatment with losartan can engender changes in cardiac and vascular parameters similar to those previously observed with ACE inhibitors.
Male SHRSP bred at the Department of Pharmacology in Heidelberg since 1975 and male Wistar rats (Mollegard, Skensved, Denmark) were used. All rats were housed at constant humidity (60±5%) and temperature (25±1°C) and kept on a 12-hour light/dark cycle throughout the duration of the experiment. The study was performed in accordance with the guidelines for animal experiments of the University of Heidelberg and was approved by the German governmental office dealing with animal protection.
In experiment 1, SHRSP were treated in utero and subsequently up to 20 weeks of age with the AT1 receptor antagonist losartan at a dose of 30 mg/kg per day (n=7). Control rats received vehicle (distilled water) (n=7). During pregnancy (3 weeks) and lactation (4 weeks), losartan or vehicle was applied to the mothers by overnight drinking water. The losartan dose was adjusted every second to third day according to the drinking habits and body weights of the mothers. After separation from their mothers, pups continued to be treated by addition of the drug to the overnight drinking water, with careful adjustment to the individual drinking habits of the growing rats. Body weight and fluid uptake were measured every second to third day.
In experiment 2, Wistar rats were treated for 6 weeks with losartan at an oral dose of 30 mg/kg per day (overnight drinking water) (n=8). Control rats received vehicle (distilled water) (n=8). Treatment with losartan or vehicle was started at 14 weeks of age.
Blood Pressure Measurement
In experiment 1, blood pressure was measured by tail plethysmography with rats under light ether anesthesia at 2-week intervals. Measurements were begun when the rats were 6 weeks old.
In experiment 2, at the end of the 6-week treatment period, rats were anesthetized and an arterial catheter (PP10 in PP50) was inserted into the right femoral artery and brought out at the back of the neck. Twenty-four hours later, the catheter was connected to a pressure transducer (Statham P23 Db, Schubart) and an eight-channel recorder (Hellige GmbH) for direct measurement of mean arterial blood pressure in conscious rats.
Isolated Rat Heart Preparation
At the end of the treatment period, rats were anesthetized with 200 mg/kg hexobarbital IP, and the hearts were removed and perfused with Krebs-Henseleit buffer of the following composition (mmol/L): NaCl 113.8, NaHCO3 22, KCl 4.7, KH2PO4 1.2, MgSO4 1.1, CaCl2 2.5, glucose 11, and sodium pyruvate 2. The solution was continuously gassed with 95% O2/5% CO2 for adjustment to pH 7.4 and was maintained at 37°C. The perfusate did not recirculate, and the hearts were not stimulated. A coronary perfusion pressure of 64 mm Hg was held constant during the experiment. After a 20-minute equilibration period, a silicone balloon was placed in the left ventricle, closely fitting the ventricular cavity, and was connected to an artificial systemic circulation. Pressure-volume work was performed by ejection of saline from the balloon into the separate artificial circulation. Cardiac output direction was regulated by two valves, and pulsatile outflow was damped by a Windkessel in the afterload side. Afterload and preload were held constant at 65 and 7.4 mm Hg, respectively. Thus, coronary and systemic circulations were completely separated, and we could measure intraventricular pressure without damaging the heart muscle.12
Determination of Cardiodynamic Parameters
Left ventricular pressure was measured by a pressure transducer (Statham P23 Db) that, on differentiation, yielded dP/dtmax and heart rate. Coronary flow was determined by an electromagnetic flow probe in the aortic cannula. All parameters were recorded on a Gould-Brush 2600 recorder (Schubart).12 Data presented are mean values over a 20-minute perfusion period.
Determination of Intracellular Parameters in the Venous Effluent
For determination of lactate release, LDH, and creatine kinase activities in the perfusate, samples were taken from the coronary effluent and analyzed spectrophotometrically.13 14 15 LDH and creatine kinase activities are expressed as milliunits per minute per gram wet weight. Wet weight of the heart was determined immediately after excision before perfusion. Lactate concentrations in the perfusate are reported as micromoles per minute per gram wet weight.
Determination of Metabolic Parameters in Myocardial Tissue
After the experiments, left ventricles were rapidly frozen in liquid nitrogen. Part of the left ventricle (approximately 500 mg) was transferred into 5 mL of 0.6N HClO4 and homogenized (Ultra-Turrax). Glycogen was hydrolyzed to glucose with amyloglycosidase (pH 4.8).16 Lactate was analyzed spectrophotometrically.13 ATP and creatine phosphate in the myocardial tissue were measured as described previously.17 18 Myocardial tissue parameters are expressed as micromoles per gram dry weight. Left ventricular weight was determined as the parameter for cardiac LVH.
Aortic cGMP Content in SHRSP
At the end of the treatment period, the thoracic aorta was excised, dissected, and snap-frozen in liquid nitrogen. The tissue was pulverized by a cell disrupter and transferred into 1 mol/L formic acid/acetone (15:85, vol/vol). After centrifugation, an aliquot of the supernatant was analyzed for cGMP with a radioimmunoassay kit (DuPont de Nemours, NEN Division). Protein determinations were performed according to Lowry et al.19
Losartan potassium (2-n-butyl-4-chloro-5-hydroxymethyl-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole, potassium salt) was obtained from DuPont Merck.
Data are reported as mean±SE. Statistical analysis between groups was performed by unpaired Student's t test when appropriate. Statistics on cardiac parameters were done on the original data. Analysis of water intake data and body weight data was performed by ANOVA with repeated measures. When a significant difference was indicated between groups, a univariate F test was used for analysis of differences between the control and losartan groups. A value of P<.05 was accepted as statistically significant.
Effect of Chronic AT1 Receptor Blockade on Body Weight and Water Intake
The development of body weight and the average water intake in losartan- and vehicle-treated SHRSP are shown in Table 1⇓. During the first 9 weeks of age, growth of losartan-treated SHRSP was slightly but not significantly delayed compared with vehicle-treated rats. On the other hand, losartan-treated SHRSP showed an increased water intake up to an age of 10 weeks (P<.05). Thereafter, body weight and water intake were similar in both groups. At 20 weeks of age, water intake decreased (P<.05) and body weight increased (P=NS) in losartan-treated compared with vehicle-treated rats.
Effect of Chronic AT1 Receptor Blockade on Blood Pressure and Left Ventricular Weight
Blood pressure development in SHRSP is shown in Fig 1⇓. In vehicle-treated rats, the major increase in blood pressure occurred between 6 and 10 weeks of age. Blood pressure further increased up to the age of 16 weeks, reaching a plateau thereafter. Oral treatment of SHRSP prenatally and subsequently up to 20 weeks of age with losartan at 30 mg/kg per day delayed and markedly attenuated the development of hypertension (Fig 1⇓). Losartan prevented the development of LVH in SHRSP, as demonstrated by a significant reduction in left ventricular weight compared with vehicle-treated rats (Fig 1⇓).
In normotensive Wistar rats, oral treatment with losartan for 6 weeks caused a slight but nonsignificant reduction in mean arterial blood pressure compared with vehicle-treated rats (99±6 versus 109±5 mm Hg in vehicle-treated rats).
Effect of Chronic AT1 Receptor Blockade on Cardiac Function and Metabolism
At the end of the 20-week treatment period, cardiac parameters were measured ex vivo in isolated rat heart preparations. Long-term oral treatment of SHRSP with losartan increased the contractility of the isolated hearts, as demonstrated by an increase in left ventricular pressure (22.4%) and dP/dtmax (35.1%). Coronary flow was increased by 38% in hearts from losartan-treated rats compared with hearts from vehicle-treated rats, whereas heart rate was unaltered (Fig 2⇓).
In the coronary venous effluent of vehicle-treated rats, intracellular components such as LDH, creatine kinase, and lactate were traced, demonstrating a certain degree of cell damage during the incubation period. The procedure of isolation of the hearts produces a short-lasting and weak ischemia, followed by reperfusion after connection of the hearts to the perfusion system. In hearts from losartan-treated rats, the activities of LDH and creatine kinase as well as the concentration of lactate in the coronary effluent were reduced by 46.4%, 47.2%, and 63.6%, respectively (Fig 2⇑).
In losartan-treated SHRSP, myocardial tissue levels of glycogen, ATP, and creatine phosphate were increased by 43.2%, 33.1%, and 42.4%, respectively, whereas lactate was decreased by 57.0% (Fig 2⇑).
Oral treatment of normotensive Wistar rats for 6 weeks with 30 mg/kg per day losartan had no effect on cardiodynamic parameters and did not affect LDH and creatine kinase enzyme activities or lactate concentration in the venous effluent as well as myocardial concentrations of glycogen, ATP, creatine phosphate, and lactate (Fig 3⇓).
Basal levels of LDH and creatine kinase activities and lactate concentration in the venous effluent were higher in hearts from vehicle-treated SHRSP compared with hearts from vehicle-treated Wistar rats (P<.05) (Table 2⇓). Thus, untreated hypertrophic hearts are less able to compensate the above-mentioned weak ischemia and reperfusion stress inherent to the preparation. In addition, left ventricular pressure, dP/dtmax, and coronary flow as well as the myocardial tissue concentrations of glycogen, ATP, and creatine phosphate were lower in SHRSP compared with Wistar rats (P<.05) (Table 2⇓).
Effect of Chronic AT1 Receptor Blockade on Aortic cGMP Content
Early-onset chronic AT1 receptor blockade increased aortic cGMP content in SHRSP nearly fivefold compared with control rats (Fig 4⇓).
The present study demonstrates that long-term blockade of AT1 receptors with losartan in SHRSP exerts a beneficial action on cardiac function and metabolism. Myocardial contractility and coronary flow were increased; the release of the intracellular enzymes LDH and creatine kinase into the coronary effluent was reduced; myocardial tissue concentrations of glycogen, ATP, and creatine phosphate were increased; and myocardial lactate concentration was decreased. This pattern of change in cardiodynamic and metabolic parameters is strikingly similar to the one previously observed after long-term treatment with the ACE inhibitor ramipril at a comparably antihypertensive dose.9 However, it is important to note that in our previous study, the cardiac effects of the ACE inhibitor were abolished after cotreatment with the B2 receptor antagonist icatibant. This observation led us to the conclusion that the cardiac effects of the ACE inhibitor depended on its inhibitory action on bradykinin breakdown and that the inhibition of Ang II generation did not play a major role in this respect. In the light of these previous findings, it is difficult to interpret the cardiac effects of the AT1 receptor antagonist observed in the present study. A number of possibilities can be considered. First, the beneficial effects of losartan on cardiac function and metabolism might have been the result of the prevention of hypertension and LVH. In the present study, cardiac performance in adult SHRSP with LVH was found to be markedly impaired compared with that in normotensive Wistar rats without LVH. Losartan had not affected cardiac performance in nonhypertrophied hearts from normotensive Wistar rats. Thus, we can speculate that losartan, just by preventing LVH, one of the most important factors for the development of myocardial dysfunction, may have improved cardiac performance.
On the other hand, a qualitatively and quantitatively similar pattern of change in cardiac parameters was also observed after long-term treatment with a low dose of the ACE inhibitors ramipril and perindopril, which had no effect on the development of hypertension and cardiac hypertrophy.9 10 Like the effects of the antihypertensive ACE inhibitor doses, these effects were also antagonized by chronic blockade of B2 receptors. Therefore, at least in the case of ACE inhibitors, the cardiac actions of renin-angiotensin system inhibition with concomitant bradykinin potentiation appear to be independent of the antihypertensive and antihypertrophic effects.
Second, there are some differences in the inhibitory actions of ACE inhibitors and AT1 receptor antagonists on the renin-angiotensin system. Although ACE inhibitors attenuate Ang II generation, AT1 receptor antagonists selectively block the AT1 receptor–mediated actions of Ang II. Since the negative feedback control of renin release is interrupted by AT1 blockade, treatment with AT1 receptor antagonists is associated with marked increases in plasma and tissue concentrations of Ang II.20 In a recent study, Campbell et al20 demonstrated a 25-fold increase in plasma Ang II concentration after 2 weeks of losartan administration (10 mg/kg BID). Furthermore, Ang II levels in heart and aortic tissue were increased by 2.4- and 14-fold, respectively. One important consequence of the losartan-induced increase in Ang II levels is the stimulation of angiotensin receptors other than the AT1 receptors. The AT2 receptor has been cloned and biochemically characterized,21 22 but there is still some debate with regard to its functional importance.23 24 25 This angiotensin receptor may be involved in fetal development and differentiation because it is highly expressed in fetal and embryonic tissues. Furthermore, recent studies have suggested that AT2 receptors exert antiproliferative effects on cultured endothelial cells.26 27 In addition, there is some evidence that stimulation of endothelial AT2 receptors is associated with the release of NO (see below).
AT2 receptors have been localized in cardiac tissues of different species. In rats, AT1 and AT2 receptors are widely and equally distributed throughout the heart, as evidenced by in situ autoradiographic binding studies.28 Suzuki et al29 reported a threefold increase in the mRNA of the AT1A receptor and a twofold increase in overall Ang II receptor density in hypertrophied hearts of spontaneously hypertensive rats and two-kidney, one clip hypertensive rats. The AT1-AT2 ratio was unaltered during hypertrophy. Lopez et al30 suggested downregulation of AT1 receptors during hypertrophy, with a redistribution of angiotensin receptors. The ratio of AT1 to AT2 of 69 to 31 in normal rats changed to 40 to 60 in hypertrophic hearts from rats with aortic banding. Thus, AT2 receptors are present in the heart and may gain more importance during the development of cardiac hypertrophy. The functional significance of the AT2 receptor in the heart is still obscure; most of the cardiac effects of Ang II appear to be AT1 mediated.31
A number of recent studies have suggested the existence of a link between Ang II and the NO system. In bovine aortic endothelial cells, Ang II (10−7 to 10−4 mol/L) dose-dependently stimulated cGMP production.32 More recently, Seyedi et al33 demonstrated NO production by measuring nitrite release of coronary microvessels and coronary arteries in response to a number of angiotensin peptides, including Ang II (10−5 mol/L). In both studies, the effects of Ang II were attenuated by NO synthase inhibition and B2 receptor blockade, suggesting that Ang II can induce NO release by activation of local kinin production. Whether these effects of Ang II were mediated by stimulation of the AT1 or AT2 receptor has not been clearly established.
In isolated rat hearts subjected to postischemic reperfusion arrhythmias, perfusion with the AT1 receptor antagonist S0029 reduced the incidence and duration of ventricular fibrillation.32 A possible link between AT1 receptor antagonism and the bradykinin and NO system is suggested by the finding that B2 receptor blockade or NO synthase inhibition had prevented the effects of S0029 in this isolated rat heart model.
In addition to the effects on cardiac function and metabolism, long-term losartan treatment in SHRSP increased aortic cGMP levels by almost fivefold compared with vehicle-treated SHRSP. This effect of chronic AT1 receptor blockade on aortic cGMP content was even more marked than the one produced by equally antihypertensive chronic ACE inhibitor treatment, as shown previously.11 In our previous study, we had shown that long-term ramipril treatment with an antihypertensive dose of 1 mg/kg per day increased aortic cGMP content 2.6-fold. This effect of the ACE inhibitor was abolished by B2 receptor blockade and could thus be ascribed to bradykinin potentiation. Like the cardiac actions, the vascular effects of losartan and ramipril may be partly ascribed to the prevention of hypertension, since high blood pressure has been shown to be associated with functional changes of the vascular wall. In particular, endothelium-dependent relaxation of blood vessels in response to different agonists has been shown to be impaired in hypertensive compared with normotensive animals.34 35 36 37
Several possibilities have to be considered to explain the effect of losartan on vascular cGMP content. First, increased Ang II levels induced by AT1 receptor blockade can stimulate non-AT1 receptors, such as the AT2 receptor, as outlined above. An involvement of a non–AT1 receptor mechanism in the stimulation of guanylate cyclase in aortic tissue has been described previously.38 In this previous study, Ang II stimulated cGMP production in incubated aortic segments. This effect was blocked by an NO synthase inhibitor and an inhibitor of soluble guanylate cyclase but not by losartan.38
Second, a direct effect of losartan on aortic ACE expression and activity has been suggested.39 In rats with aortic coarctation, 2-week administration of losartan at a sub-antihypertensive dose of 3 mg/kg per day reduced ACE expression and ACE activity to the level of sham-operated animals, a mechanism that might also result in decreased bradykinin breakdown. Although losartan does not appear to alter plasma and tissue levels of bradykinin after short-term treatment of normotensive rats,20 it remains to be shown whether it can increase bradykinin levels in hypertensive rats and after long-term administration.
In conclusion, chronic blockade of AT1 receptors with losartan improved cardiac function and metabolism and increased aortic cGMP content in SHRSP to an extent similar to or even greater than that observed previously after long-term ACE inhibitor treatment. The prevention of hypertension and cardiac and vascular hypertrophy as well as the stimulation of non-AT1 receptors, such as the AT2 receptor, may be involved in the mechanism of action of losartan. Further studies are needed to elucidate the exact mechanisms underlying the cardioprotective effects of losartan in hypertension-induced LVH.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|AT1, AT2||=||angiotensin type 1, type 2|
|dP/dtmax||=||differentiated left ventricular pressure|
|LVH||=||left ventricular hypertrophy|
|SHRSP||=||stroke-prone spontaneously hypertensive rat(s)|
- Received September 22, 1995.
- Revision received November 29, 1995.
- Accepted April 4, 1996.
Linz W, Wiemer G, Gohlke P, Unger T, Schölkens BA. Contribution of kinins to the cardiovascular actions of angiotensin-converting enzyme inhibitors. Pharmacol Rev. 1995;47:25-49.
Liu Y-H, Yang X-P, Sharov VG, Sigmon DH, Sabbah HN, Carretero OA. Paracrine systems in the cardioprotective effect of angiotensin-converting enzyme inhibitors on myocardial ischemia/reperfusion injury in rats. Hypertension. 1996;27:7-13.
Gasparo M, Husain A, Alexander W, Catt KJ, Chiu AT, Frew M, Goodfriend T, Harding JW, Inagami T, Timmermans PBMWM. Proposed update of angiotensin receptor nomenclature. Hypertension. 1995;25:924-927.
Camargo MJF, Von Lutterotti N, Campbell WG Jr, Pecker MS, James GD, Timmermans PBMWM, Laragh JH. Control of blood pressure and end-organ damage in maturing salt-loaded stroke prone spontaneously hypertensive rats by oral angiotensin II receptor blockade. J Hypertens. 1993;11:31-40.
Stier CT Jr, Adler LA, Levine S, Chander PN. Stroke prevention by losartan in stroke-prone spontaneously hypertensive rats. J Hypertens. 1993;11(suppl 3):S37-S42.
Stier CTJ, Benter IF, Ahmad S, Zuo H, Selig N, Roethel S, Levine S, Itskovitz HD. Enalapril prevents stroke and kidney dysfunction in salt-loaded stroke-prone spontaneously hypertensive rats. Hypertension. 1989;13:115-121.
Gohlke P, Linz W, Schölkens BA, Kuwer I, Bartenbach S, Schnell A, Unger T. Angiotensin converting enzyme inhibition improves cardiac function: role of bradykinin. Hypertension. 1994;23:411-418.
Gohlke P, Lamberty V, Kuwer I, Bartenbach S, Schnell A, Linz W, Schölkens BA, Wiemer G, Unger T. Long-term low-dose angiotensin converting enzyme inhibitor treatment increases vascular cyclic guanosine 3′,5′-monophosphate. Hypertension. 1993;22:682-687.
Linz W, Schölkens BA, Han YF. Beneficial effects of the converting enzyme inhibitor, ramipril, in ischemic rat hearts. J Cardiovasc Pharmacol. 1986;8(suppl 10):S91-S99.
Noll F. L-(+)-Lactate. In: Bergemeyer HU, Bergemeyer J, Grassl M, eds. Methods of Enzymatic Analysis. Metabolites I: Carbohydrates. Deerfield Beach, Fla: Weinheim/Basel, Switzerland: Verlag Chemie; 1984;6:582-588.
Vassault A. Lactate dehydrogenase. UV-method with pyruvate and NADH. In: Bergemeyer HU, Bergemeyer J, Grassl M, eds. Methods of Enzymatic Analysis. Enzymes 1: Oxidoreductases, Transferases. Deerfield Beach, Fla: Weinheim/Basel, Switzerland: Verlag Chemie; 1983;13:118-126.
Gerhardt W. Creatine kinase. Routine UV-method. In: Bergemeyer HU, Bergemeyer J, Grassl M, eds. Methods of Enzymatic Analysis. Enzymes 1: Oxidoreductases, Transferases. Deerfield Beach, Fla: Weinheim/Basel, Switzerland: Verlag Chemie; 1983;3:510-518.
Keppler D, Decker K. Glycogen. In: Bergemeyer HU, Bergemeyer J, Grassl M, eds. Methods of Enzymatic Analysis. Metabolites 1: Carbohydrates. Deerfield Beach, Fla: Weinheim/Basel, Switzerland: Verlag Chemie; 1984;6:11-18.
Trautschold I, Lamprecht W, Schweitzer G. Adenosine-5-triphosphate. UV-method with hexokinase and glycose-6-phosphate dehydrogenase. In: Bergemeyer HU, Bergemeyer J, Grassl M, eds. Methods of Enzymatic Analysis. Metabolites 2: Tri- and Dicarboxylic Acids, Purines, Pyrimidines and Derivates, Coenzymes, Inorganic Compounds. Deerfield Beach, Fla: Weinheim/Basel, Switzerland: Verlag Chemie; 1985;7:346-357.
Heinz F, Weisser H. Creatine phosphate. In: Bergemeyer HU, Bergemeyer J, Grassl M, eds. Methods of Enzymatic Analysis. Metabolites 3: Lipids, Amino Acids and Related Compounds. Deerfield Beach, Fla: Weinheim/Basel, Switzerland: Verlag Chemie; 1985;8:507-514.
Lowry OH, Rosebrough NJ, Farr AL, Randall R. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.
Kambayashi Y, Bardhan S, Takahashi K, Tsuzuki S, Inui H, Hamakubo T, Inagami T. Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine phosphatase inhibition. J Biol Chem. 1993;268:24543-24546.
Mukoyama M, Nakajima M, Horiuchi M, Sasamura H, Pratt RE, Dzau VJ. Expression cloning of type 2 angiotensin II receptor reveals a unique class of seven-transmembrane receptors. J Biol Chem. 1993;268:24539-24542.
De Gasparo M, Levens NR, Kamber B, Furet P, Whitebread S, Brechler V, Bottari SP. The angiotensin II AT2 receptor subtype. In: Saavedra JM, Timmermans PB, eds. Angiotensin Receptors. New York, NY: Plenum Press; 1994:95-117.
Metsärinne KP, Stoll M, Gohlke P, Paul M, Unger T. Angiotensin II is antiproliferative for coronary endothelial cells in vitro. Pharm Pharmacol Lett. 1992;2:150-152.
Stoll M, Steckelings UM, Paul M, Bottari SP, Metzger R, Unger T. The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells. J Clin Invest. 1995;95:651-657.
Sechi LA, Griffin CA, Grady EF, Kalinyak JE, Schambelan M. Characterization of angiotensin II receptor subtypes in rat heart. Circ Res. 1992;71:1482-1489.
Suzuki J, Matsubara H, Urakami M, Inada M. Rat angiotensin II (type 1A) receptor mRNA regulation and subtype expression in myocardial growth and hypertrophy. Circ Res. 1993;73:439-447.
Lopez JJ, Lorell BH, Ingelfinger JR, Weinberg EO, Schunkert H, Diamant D, Tang SS. Distribution and function of cardiac angiotensin AT1 and AT2 receptor subtypes in hypertrophied rat hearts. Am J Physiol. 1994;267:H844-H852.
Timmermans PB, Smith RD. Angiotensin II receptor subtypes: selective antagonists and functional correlates. Eur Heart J. 1994;15:79-87.
Wiemer G, Schölkens BA, Busse R, Wagner A, Heitsch H, Linz W. The functional role of angiotensin II, subtype AT2, receptors in endothelial cells and isolated ischemic rat hearts. Pharm Pharmacol Lett. 1993;3:24-27.
Seyedi N, Xu XB, Nasjiletti A, Hintze TH. Coronary kinin generation mediates nitric oxide release after angiotensin receptor stimulation. Hypertension. 1995;26:164-170.
Lüscher TF, Vanhoutte PM. Endothelium-dependent contractions to acetylcholine in the aorta of the spontaneously hypertensive rat. Hypertension. 1986;8:344-348.
Shimamura K, Osugi S, Moiyama K, Sunano S. Impairment and protection of endothelium-dependent relaxation in aortae of various strains of spontaneously hypertensive rats. J Cardiovasc Pharmacol. 1991;17(suppl 3):S133-S136.
Shirasaki Y, Kolm P, Nickols GA, Lee TJ-F. Endothelial regulation of cyclic GMP and vascular responses in hypertension. J Pharmacol Exp Ther. 1988;245:53-58.
Munzenmaier DH, Greene AS. Stimulation of soluble guanylate cyclase activity by angiotensin II is mediated by a non-AT1 receptor mechanism in rat aorta. FASEB J. 1994;8:A367. Abstract.
Holtz J, Munzel T, Sommer O, Bassenge E. Converting enzyme inhibition by enalapril in experimental heart failure. Nephron. 1990;55(suppl 1):73-76.