J Mol Cell Cardiol
Effect
23, 665-670
of Captopril
Mitsuyuki Kazuhiko
(1991)
on Isoproterenol-induced Decarboxylase Activity
Shimizu, Osamu Irimajiri, Tomoko Ogawa, Junk0 Sanjo, Han&a Yamada, Isogai
Myocardial
Nakano, Hideki
Ornithine
Tsuneo Mizokami, Sasaki and Yukihide
Third Department of Internal Medicine, The Jikei University School of Medicine, Nshi-shinbashi 3-25-8, Minato-ku, Tokyo, Japan (Received 10 October 1989, accepted in revisedform
11 September 1990)
hl. SHIMIZU, 0. IRIMAJIRI, T. NAKANO, T. MIZOKAMI, K. OGAWA, J. SANJO, H. YAMADA, H. SASAKI AND Y. ISOCAI, Effect ofcaptopril on Isoproterenol-induced Myocardial Ornithine Decarboxylase Activity. ,~oournal oJ‘ Moleculnr and Cellular Cardiology (1991) 23, 665-670. Polyamines are thought to play an essential role in cellular hypertrophy and proliferation. Ornithine decarboxylase (ODC) catalyzes the first and probably the ratrlimiting step in biosynthesis of polyamines. In this study, we evaluated the pathophysiological role of the reninangiotensin system in isoproterenol-induced cardiac hypertrophy, using myocardial ODC activity as an indicator of cardiac hypertrophy. Isoproterenol caused an eight-fold increase of myocardial ODC activity in normotensive Wistar rats within 4 h after injection. Captopril suppressed the induction ofODC by isoproterenol to two-thirds of the control level. These results indicate that the renin-angiotensin system may participate in the induction of myocardial hypertrophy by isoproterenol. KEY WORDS: hypertrophy.
Rat;
Ornithine
decarboxylase;
Isoproterenol;
Introduction Cardiac hypertrophy has been attributed to various factors besides sustained hypertension [6, 81. For example, the renin-angiotensin system is presumed to play an important role in the development of hypertrophy, althoygh the detailed mechanism is not yet known [5, 15, 221. Several studies have suggested that angiotensin II may contribute to myocardial hypertrophy by direct stimulation of myocardial protein synthesis [IO, 241. Isoproterenol is known to activate the renin-angiotensin system by stimulating the p-receptor [9], and to induce cardiac hypertrophy [31]. Putrescine, spermidine and spermine, which are generally known as polyamines, increase during cellular growth and hypertrophy and are presumed to play an essential role in these processes [7, 19, 241. Ornithine decarboxylase (ODC) is the key enzyme in the pathway for polyamine biosynthesis. Its activity exhibits a dramatic increase following various types of growth stimuli, an extremely short half-life (11 to 20 min) [19,25]. In the present study, myocardial OOZZ-2828/91/060665
+ 6 $03.00/O
Captopril;
Renin-angiotensin
system;
Cardiac
ODC activity was used as a marker of cardiac hypertrophy and the participation of the renin-angiotensin system in isoproterenolinduced cardiac hypertrophy was examined using captopril, which is an angiotensinconverting enzyme inhibitor. Materials
and
Experimental
Methods animals
Nine-week-old SHR, WKY and normotensive Wistar rats derived from colonies maintained at the Department of Pharmacology of Jichi Medical School were used. They were purchased from the Hoshino Laboratory Animal Co., Inc. Rats were maintained for more than 1 week in a controlled environment (temperature, 22 + 1°C; humidity, 55 f 5”{,; lighting time from 9:00 to 21:00 h). They were fed commercial laboratory chow ad libitum. Determination
of myocardial ODC activify
Hearts were excised, cut into small pieces with scissors in 5 ml Of a SOlUtiOn COnSiSting of0.25 M 0 1991 Academic
Press Limited
666
M.
Shimizu
sucrose and 1 mM dithiothreitol, and homogenized using a polytoron. The homogenate was centrifuged at 8000 x g for 30 min, and the supernatant was assayed for ODC activity in an Eppendorf tube using the method of Murakami et al. [ISJ. One unit of enzyme activity was defined as the amount catalyzing the formation of 1 nmol of CO per hour. Determintion
of myocardial po&amine concentrations
Hearts were homogenized with 5 ml of 0.25 M sucrose and then treated with 2q/, perchloric acid. After centrifugation at 8000 x g for 30 min, the supernatant was analyzed by highperformance liquid chromatography (Shimazu, model LC-3A) performed with an LC column (ISC-05/so 504). Chromatography was performed at 70°C with a flow rate of 0.7 ml/min using 0.16 M sodium citrate and 1.6 M NaCl. The eluate was mixed with 6 mM o-phthaladehyde dissolved in a solution containing 0.38 M Na,CO,, 0.108 M KzSO,, 0.22 0. lyO Briji 35, and 2.9 mM 2M HJQ, mercaptoethanol, and then passed through the flow cell of a fluoro-monitor at 0.5 ml/min.
Determination of protein and nucleic acids The protein concentration was measured by the Biuret method [12]. Nucleic acids were extracted by Schneider’s method [271 and the color was developed with diphenylamine (DNA) or orcinol (RNA).
Statistical analysis Results are expressed as the mean f S.D. Statistical significance was established using Student’s t-test.
Results Effect of isoproterenol on myocardial ODC activity, polyamine concentrations and nucleic acid content The effect of a single subcutaneous injection of isoproterenol (20 mg/kg) on the various parameters of cardiac hypertrophy is shown in Figure 1. A stimulatory effect of isoproterenol on ODC activity was seen from 4 h after treatment, and the peak rise in the enzyme
et al.
activity occurred at 8 h putrescine concentration marked increase, reaching treatment. Spermidine ually increased, whereas tion gradually decreased ment. Both the RNA increased gradually until
after injection. ‘l’hr also showed iI a peak at 12 h afirr concentration gradspermine concentrauntil 16 h after treatand DNA content 16 h after treatment.
Effect of a single oral dose of captopril on qocardial polyamine concentrations in SHR It has been reported that there are marked circadian changes in hepatic and renal polyamine concentrations in rats, with a peak at around 22:00 h [17, 281. Therefore, circadian changes need to be considered in any study of tissue polyamine levels. Accordingly, captopril was administered to SHR rats by gastric tube at 16:00 h, and myocardial polyamine levels were measured at 16:00, 19:00 and 22:00 h. As shown in Figure 2, captopril significantly suppressed the circadian increase of cardiac spermidine (432 f 33 vs. 355 _+ 56 nmol/mg DNA), but not that of spermine. Effect of captopril on isoproterenol-induced m-yocardial ODC activity Normotensive rats were divided into four groups. Group I was sacrificed at zero time without any treatment. Group II was subcutaneously injected with saline and sacrificed 4 h later. Group III was injected with isoproterenol (20 mg/kg) and sacrificed 4 h later. Group IV was similar to group III, except that the rats received captopril orally by gastric tube (30 mg/kg) 1 h before the isoprotereno1 injection. As shown in Figure 3, ODC activity showed approximately an eight-fold increase in the isoproterenol-treated group, when compared with the saline-treated group. Administration of captopril 1 h before isoproterenol suppressed the induction of ODC to two-thirds of the control level. Discussion The biosynthesis of myocardial proteins is accelerated during sustained hypertension [S, S]. To study the pathogenesis of cardiac hypertrophy, it is essential to assess not only
Effect
4
8
667
of Captopril
12
I6 Time
Ih)
FIGURE I. Effect ofisoproterenol on myocardial ODC activity, polyamine -- -, untreated rats; ---, isoproterenol-treated rats; n = 5. Rats were isoproterenol (20 mg/kg) at zero time. Values are the means z!c S.D. *, ** (*P
Effect
23, 665-670
of Captopril
Mitsuyuki Kazuhiko
(1991)
on Isoproterenol-induced Decarboxylase Activity
Shimizu, Osamu Irimajiri, Tomoko Ogawa, Junk0 Sanjo, Han&a Yamada, Isogai
Myocardial
Nakano, Hideki
Ornithine
Tsuneo Mizokami, Sasaki and Yukihide
Third Department of Internal Medicine, The Jikei University School of Medicine, Nshi-shinbashi 3-25-8, Minato-ku, Tokyo, Japan (Received 10 October 1989, accepted in revisedform
11 September 1990)
hl. SHIMIZU, 0. IRIMAJIRI, T. NAKANO, T. MIZOKAMI, K. OGAWA, J. SANJO, H. YAMADA, H. SASAKI AND Y. ISOCAI, Effect ofcaptopril on Isoproterenol-induced Myocardial Ornithine Decarboxylase Activity. ,~oournal oJ‘ Moleculnr and Cellular Cardiology (1991) 23, 665-670. Polyamines are thought to play an essential role in cellular hypertrophy and proliferation. Ornithine decarboxylase (ODC) catalyzes the first and probably the ratrlimiting step in biosynthesis of polyamines. In this study, we evaluated the pathophysiological role of the reninangiotensin system in isoproterenol-induced cardiac hypertrophy, using myocardial ODC activity as an indicator of cardiac hypertrophy. Isoproterenol caused an eight-fold increase of myocardial ODC activity in normotensive Wistar rats within 4 h after injection. Captopril suppressed the induction ofODC by isoproterenol to two-thirds of the control level. These results indicate that the renin-angiotensin system may participate in the induction of myocardial hypertrophy by isoproterenol. KEY WORDS: hypertrophy.
Rat;
Ornithine
decarboxylase;
Isoproterenol;
Introduction Cardiac hypertrophy has been attributed to various factors besides sustained hypertension [6, 81. For example, the renin-angiotensin system is presumed to play an important role in the development of hypertrophy, althoygh the detailed mechanism is not yet known [5, 15, 221. Several studies have suggested that angiotensin II may contribute to myocardial hypertrophy by direct stimulation of myocardial protein synthesis [IO, 241. Isoproterenol is known to activate the renin-angiotensin system by stimulating the p-receptor [9], and to induce cardiac hypertrophy [31]. Putrescine, spermidine and spermine, which are generally known as polyamines, increase during cellular growth and hypertrophy and are presumed to play an essential role in these processes [7, 19, 241. Ornithine decarboxylase (ODC) is the key enzyme in the pathway for polyamine biosynthesis. Its activity exhibits a dramatic increase following various types of growth stimuli, an extremely short half-life (11 to 20 min) [19,25]. In the present study, myocardial OOZZ-2828/91/060665
+ 6 $03.00/O
Captopril;
Renin-angiotensin
system;
Cardiac
ODC activity was used as a marker of cardiac hypertrophy and the participation of the renin-angiotensin system in isoproterenolinduced cardiac hypertrophy was examined using captopril, which is an angiotensinconverting enzyme inhibitor. Materials
and
Experimental
Methods animals
Nine-week-old SHR, WKY and normotensive Wistar rats derived from colonies maintained at the Department of Pharmacology of Jichi Medical School were used. They were purchased from the Hoshino Laboratory Animal Co., Inc. Rats were maintained for more than 1 week in a controlled environment (temperature, 22 + 1°C; humidity, 55 f 5”{,; lighting time from 9:00 to 21:00 h). They were fed commercial laboratory chow ad libitum. Determination
of myocardial ODC activify
Hearts were excised, cut into small pieces with scissors in 5 ml Of a SOlUtiOn COnSiSting of0.25 M 0 1991 Academic
Press Limited
666
M.
Shimizu
sucrose and 1 mM dithiothreitol, and homogenized using a polytoron. The homogenate was centrifuged at 8000 x g for 30 min, and the supernatant was assayed for ODC activity in an Eppendorf tube using the method of Murakami et al. [ISJ. One unit of enzyme activity was defined as the amount catalyzing the formation of 1 nmol of CO per hour. Determintion
of myocardial po&amine concentrations
Hearts were homogenized with 5 ml of 0.25 M sucrose and then treated with 2q/, perchloric acid. After centrifugation at 8000 x g for 30 min, the supernatant was analyzed by highperformance liquid chromatography (Shimazu, model LC-3A) performed with an LC column (ISC-05/so 504). Chromatography was performed at 70°C with a flow rate of 0.7 ml/min using 0.16 M sodium citrate and 1.6 M NaCl. The eluate was mixed with 6 mM o-phthaladehyde dissolved in a solution containing 0.38 M Na,CO,, 0.108 M KzSO,, 0.22 0. lyO Briji 35, and 2.9 mM 2M HJQ, mercaptoethanol, and then passed through the flow cell of a fluoro-monitor at 0.5 ml/min.
Determination of protein and nucleic acids The protein concentration was measured by the Biuret method [12]. Nucleic acids were extracted by Schneider’s method [271 and the color was developed with diphenylamine (DNA) or orcinol (RNA).
Statistical analysis Results are expressed as the mean f S.D. Statistical significance was established using Student’s t-test.
Results Effect of isoproterenol on myocardial ODC activity, polyamine concentrations and nucleic acid content The effect of a single subcutaneous injection of isoproterenol (20 mg/kg) on the various parameters of cardiac hypertrophy is shown in Figure 1. A stimulatory effect of isoproterenol on ODC activity was seen from 4 h after treatment, and the peak rise in the enzyme
et al.
activity occurred at 8 h putrescine concentration marked increase, reaching treatment. Spermidine ually increased, whereas tion gradually decreased ment. Both the RNA increased gradually until
after injection. ‘l’hr also showed iI a peak at 12 h afirr concentration gradspermine concentrauntil 16 h after treatand DNA content 16 h after treatment.
Effect of a single oral dose of captopril on qocardial polyamine concentrations in SHR It has been reported that there are marked circadian changes in hepatic and renal polyamine concentrations in rats, with a peak at around 22:00 h [17, 281. Therefore, circadian changes need to be considered in any study of tissue polyamine levels. Accordingly, captopril was administered to SHR rats by gastric tube at 16:00 h, and myocardial polyamine levels were measured at 16:00, 19:00 and 22:00 h. As shown in Figure 2, captopril significantly suppressed the circadian increase of cardiac spermidine (432 f 33 vs. 355 _+ 56 nmol/mg DNA), but not that of spermine. Effect of captopril on isoproterenol-induced m-yocardial ODC activity Normotensive rats were divided into four groups. Group I was sacrificed at zero time without any treatment. Group II was subcutaneously injected with saline and sacrificed 4 h later. Group III was injected with isoproterenol (20 mg/kg) and sacrificed 4 h later. Group IV was similar to group III, except that the rats received captopril orally by gastric tube (30 mg/kg) 1 h before the isoprotereno1 injection. As shown in Figure 3, ODC activity showed approximately an eight-fold increase in the isoproterenol-treated group, when compared with the saline-treated group. Administration of captopril 1 h before isoproterenol suppressed the induction of ODC to two-thirds of the control level. Discussion The biosynthesis of myocardial proteins is accelerated during sustained hypertension [S, S]. To study the pathogenesis of cardiac hypertrophy, it is essential to assess not only
Effect
4
8
667
of Captopril
12
I6 Time
Ih)
FIGURE I. Effect ofisoproterenol on myocardial ODC activity, polyamine -- -, untreated rats; ---, isoproterenol-treated rats; n = 5. Rats were isoproterenol (20 mg/kg) at zero time. Values are the means z!c S.D. *, ** (*P
