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(Circulation. 1999;99:817-822.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Cardioprotective Effect of Angiotensin-Converting Enzyme Inhibition Against Hypoxia/Reoxygenation Injury in Cultured Rat Cardiac Myocytes
From the Second Department of Medicine, Kyoto Prefectural University of Medicine and Department of Clinical Pharmacology (J.A.), Kyoto Pharmaceutical University, Kyoto, Japan.
Correspondence to Tetsuya Tatsumi, MD, PhD, Second Department of Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan. E-mail tatsumi{at}koto.kpu-m.ac.jp
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Abstract |
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Background—Although ACE inhibitors can protect myocardium against ischemia/reperfusion injury, the mechanisms of this effect have not yet been characterized at the cellular level. The present study was designed to examine whether an ACE inhibitor, cilazaprilat, directly protects cardiac myocytes against hypoxia/reoxygenation (H/R) injury.
Methods and Results—Neonatal rat cardiac myocytes in primary culture were exposed to hypoxia for 5.5 hours and subsequently reoxygenated for 1 hour. Myocyte injury was determined by the release of creatine kinase (CK). Both cilazaprilat and bradykinin significantly inhibited CK release after H/R in a dose-dependent fashion and preserved myocyte ATP content during H/R, whereas CV-11974, an angiotensin II receptor antagonist, and angiotensin II did not. The protective effect of cilazaprilat was significantly inhibited by Hoe 140 (a bradykinin B2 receptor antagonist), NG-monomethyl-L-arginine monoacetate (L-NMMA) (an NO synthase inhibitor), and methylene blue (a soluble guanylate cyclase inhibitor) but not by staurosporine (a protein kinase C inhibitor), aminoguanidine (an inhibitor of inducible NO synthase), or indomethacin (a cyclooxygenase inhibitor). Cilazaprilat significantly enhanced bradykinin production in the culture media of myocytes after 5.5 hours of hypoxia but not in that of nonmyocytes. In addition, cilazaprilat markedly enhanced the cGMP content in myocytes during hypoxia, and this augmentation in cGMP could be blunted by L-NMMA and methylene blue but not by aminoguanidine.
Conclusions—The present study demonstrates that cilazaprilat can directly protect myocytes against H/R injury, primarily as a result of an accumulation of bradykinin and the attendant production of NO induced by constitutive NO synthase in hypoxic myocytes in an autocrine/paracrine fashion. NO modulates guanylate cyclase and cGMP synthesis in myocytes, which may contribute to the preservation of energy metabolism and cardioprotection against H/R injury.
Key Words: angiotensin • hypoxia • bradykinin • nitric oxide • myocytes
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Introduction |
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Recent clinical and experimental studies have established the therapeutic benefits of ACE inhibitors, not only in treating hypertension and congestive heart failure, but also in reducing reinfarction, limiting infarct size, and preventing reperfusion arrhythmias.1 2 3 These cardioprotective effects of ACE inhibitors are thought to depend on their ability to attenuate the degradation of endogenous bradykinin as well as to decrease the synthesis of angiotensin II from angiotensin I. In addition to altering circulating concentrations of angiotensin II and bradykinin, thereby effecting hemodynamic changes, ACE inhibitors may participate in cardioprotection through the alteration of localized angiotensin II or bradykinin concentrations in cardiac tissue.4 Angiotensin II may induce cardiac myocyte necrosis and fibroblast proliferation, thereby exacerbating ischemia-reperfusion injury.5 In contrast, recent reports have indicated that kinin can induce an increase in coronary circulation and improve cardiac function in ischemia-reperfusion injury. Although inhibition of ACE is also known to cause coronary vasodilation and an improvement in contractile and metabolic function in ischemia-reperfusion injury,6 7 8 9 it is still unknown whether the beneficial effects of ACE inhibitors are attributable to a direct effect on cardiac myocytes.
In the present study, therefore, we focused on the direct molecular actions of an ACE inhibitor on cardiac myocytes. We have examined the following: (1) whether cilazaprilat, a nonsulfhydryl ACE inhibitor, directly protects cardiac myocytes against hypoxia/reoxygenation injury; (2) whether this protective action derives from an inhibition of angiotensin II synthesis or an accumulation of bradykinin; (3) whether this protective effect is mediated by prostaglandins, NO, or protein kinase C (PKC); and (4) whether cGMP is involved in the protective effect. We treated cultured rat neonatal cardiac myocytes with either modified Tyrode solution, cilazaprilat, angiotensin II, bradykinin, or the angiotensin II type 1 receptor antagonist CV-11974 and subsequently exposed the cells to 5.5 hours of hypoxia followed by 1 hour of reoxygenation. In addition, we tested whether pretreatment with the kinin receptor antagonist Hoe 140, the NO synthase inhibitor NG-monomethyl-L-arginine monoacetate (L-NMMA), the inducible NO synthase inhibitor aminoguanidine, the cyclooxygenase inhibitor indomethacin, or the PKC inhibitor staurosporine could block the beneficial effect of cilazaprilat. Furthermore, we measured the concentration of bradykinin in culture media after treatment with cilazaprilat and monitored the cGMP content in myocytes during hypoxia as well as the high-energy phosphates in myocytes during hypoxia/reoxygenation.
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Methods |
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Materials
Cilazaprilat, Hoe 140, and CV-11974 were generous gifts from Nippon Roch KK, Hoechst Marion Roussel AG, and Takeda Chemical Industries, Ltd, respectively. Angiotensin II and bradykinin were purchased from Peptide Institute, Inc; L-NMMA from Calbiochem-Novabiochem International; aminoguanidine from Nacalai tesque Inc; and the remaining reagents from Sigma Chemical Co.
Culture of Neonatal Rat Cardiac Myocytes
Primary cultures of neonatal rat cardiac myocytes were prepared
as previously described with some modifications.10
Briefly, hearts were removed from 1- to 2-day-old Wistar rats
anesthetized by ether under aseptic conditions and placed in
Ca2+- and Mg2+-free PBS.
The hearts were washed with PBS, and the atria and aorta were
discarded. The ventricles were minced with scissors into 1- to
3-mm3 fragments, and they were then enzymatically
digested 4 times for 10 to 15 minutes each with 7.5 mL of PBS
containing 0.2% collagenase (Sigma type I). The liberated
cells were collected by centrifugation at
300g and incubated in 100-mm culture dishes (Falcon) for 60
minutes at 37°C in a humidified incubator with 5%
CO2 air. The nonadherent cardiac myocytes were
harvested and seeded into 60-mm culture dishes
(1x105 cells/cm2). The
myocytes were incubated in Dulbecco's modified Eagle's medium (DMEM;
Nissui Pharmaceutical Co) supplemented with 10% FBS (Bioserum Co).
5-Bromo-2'-deoxyuridine (BrdU; 100 µmol/L) was added during the
first 48 hours to inhibit proliferation of
nonmyocytes.11 Using this method, we
routinely obtained contractile myocyte-rich cultures with 90% to 95%
myocytes, as assessed by immunofluorescence
staining with a monoclonal antibody against ß-myosin heavy chain. The
myocytes were then incubated in DMEM containing 0.5% FBS without BrdU,
and all experiments were done 36 to 48 hours after this
incubation.
Preparation of Cardiac Nonmyocyte-Rich Culture
Highly enriched cultures of cardiac nonmyocytes
(hereafter called nonmyocyte culture) were prepared by 2
passages of the cells adherent to the culture dish during the
preplating procedure.11 Until the second passage, cells
were maintained in the same culture medium as above, except that 10%
FBS was used and BrdU was not used. The nature of cells was determined
by immunofluorescence staining with anti-rat factor
VIII, anti-desmin, and anti-vimentin for identification of
endothelial cells, smooth muscle cells, and
fibroblasts, respectively. After the second passage, only 1% to 2% of
cells were stained positively with anti-desmin or anti-factor VIII.
More than 95% of cells were stained positively with anti-vimentin
antibody, indicating that the nonmyocytes consisted of
fibroblasts under our culture conditions.
Culture of Rat Aortic Endothelial Cells
Rat aortic endothelial cells (RAECs) were
isolated from male Wistar rats (weight, 200 to 250 g) by the
primary explant technique.12 The cells were cultured in
DMEM supplemented with 10% FBS and 0.15 mg/mL
endothelial cell growth supplement. RAECs were grown at
37°C in a humidified incubator with 5% CO2 air
and serially passaged with a split ratio of 1:5 using 0.05%
trypsin-0.02% EDTA. Biocoat Cellware (rat-tail collagen type I),
35-mm/60-mm-diameter tissue culture dishes from Becton Dickinson
Labware were used throughout the RAEC cultures. Experiments were
performed on cells at passage 3 from the primary culture.
Characterization as endothelial cells was confirmed by
immunofluorescence staining with antibodies
specific for factor VIII.
Experimental Protocols
Figure 1
shows the experimental
protocols. Before hypoxic exposure, cell medium was replaced by
modified Tyrode solution (in mmol/L: NaCl 136.9, KCl 2.68,
Na2HPO4 ·
12H2O 8.1,
KH2PO4 1.47,
CaCl2 0.9, MgCl2 ·
6H2O 0.49; pH 7.4). The cardiac myocytes were
transferred to an environmental chamber at 37°C in a humidified
atmosphere flushed with 5% CO2 and <1% oxygen
(F-102, Iijima Electronics Co) in nitrogen for 5.5 hours and were then
reoxygenated for 1 hour with DMEM.
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To examine the role of angiotensin and bradykinin in hypoxia/reoxygenation injury, the myocytes were treated with the following before hypoxia/reoxygenation: (1) modified Tyrode solution (control); (2) cilazaprilat (10-8 to 10-5 mol/L); (3) CV-11974 (10-8 to 10-5 mol/L); (4) angiotensin II (10-9 to 10-6 mol/L); and (5) bradykinin (10-9 to 10-6 mol/L). Furthermore, to examine the mechanism of the cardioprotective effect of cilazaprilat, myocytes were treated with cilazaprilat (10-5 mol/L) before hypoxia/reoxygenation in the presence of (6) Hoe 140 (10-6 mol/L), (7) staurosporine (2x10-9 mol/L), (8) L-NMMA (4x10-4 mol/L), (9) aminoguanidine (5x10-4 mol/L), (10) indomethacin (10-5 mol/L), and (11) methylene blue (10-5 mol/L).
Assay of Creatine Kinase Release
Creatine kinase (CK) activity in culture media was measured
before hypoxia, at the end of 5.5 hours of hypoxia, and
after 1 hour of reoxygenation in all groups (Figure 1
). CK activity in culture media was measured
spectrophotometrically at 37°C according to Rosalki's procedure. The
activity of CK was expressed as IU/L.13
Measurement of Bradykinin Concentration
Bradykinin concentration in the culture media was measured
during hypoxia as shown in Figure 1, according to a
previously described method.14 Briefly, 0.1-mL samples of
culture supernatant were acidified with 5 mL of 0.01 mol/L HCl and
extracted twice with 20 mL of diethyl ether. The aqueous phase was
taken to dryness with a rotary evaporator, and the dried samples were
stored at -80°C until assayed. Before assay, the dried samples were
redissolved in 2.5 mL of 0.1 mol/L Tris-HCl buffer containing 0.2%
gelatin, 0.1% neomycin, and 0.01 mol/L EDTA, adjusted to pH 7.4. The
incubation mixture for radioimmunoassay consisted of 0.1 mL of 0.01
mol/L 1,10-phenanthroline HCl, diluent buffer of 0.5 mL containing the
unknown or standard bradykinin, 0.1 mL of antiserum diluted
1:600 with diluent buffer, and 0.1 mL of
(125I-Tyr8)-bradykinin
(
8000 cpm) dissolved in normal saline. The mixture was incubated in
a polyethylene tube at 4°C for 24 hours, and dextran-coated charcoal
was used to separate the free labeled antigen from that bound to
antibody. Three replicate tubes containing only buffer, phenanthroline,
and (125I-Tyr8)-bradykinin
were incubated and treated with coated charcoal to determine the amount
of labeled antigen that remained in the supernatant in the absence of
antibody. The mean value of this measurement was subtracted from
supernatant radioactivity after centrifugation of the
antibody-containing tubes, and the resultant value was used to
calculate the proportion of label bound to antibody.
Measurement of cGMP in Cardiac Myocytes
cGMP concentration in myocytes was measured after 1, 3, and 5.5
hours of hypoxia (Figure 1). Cardiac myocytes
(2.7x106 cells per dish) were treated with 0.25
mL of ice-cold 6% trichloroacetic acid and centrifuged at
1000g for 10 minutes. The supernatant was extracted 3 times
with 3 mL of diethyl ether saturated with water, and the aqueous phase
was stored at -80°C. cGMP concentration in the supernatant was
measured by radioimmunoassay.15 Briefly, 0.1 mL of
dioxane-triethylamine mixture containing succinic acid anhydride
succinylated cGMP was added to the supernatant (0.1 mL). After a
10-minute incubation, the reaction mixture was added to 0.8 mL of 0.3
mol/L imidazole buffer (pH 6.5). Succinyl cGMP tyrosine methyl ester
(0.1 mL) iodinated with 125I (15 000
to 20 000 cpm in <10-14 mol/L) was added to
the assay mixture containing 0.1 mL of supernatant and 0.1 mL of
diluted antisera, and the mixture was incubated at 4°C for 20 hours.
A cold solution of dextran-coated charcoal (0.5 mL) was added to the
mixture in an ice-cold water bath. The charcoal was spun down, and 0.5
mL of the supernatant was counted for radioactivity in a gamma
spectrometer. The amount of cGMP was normalized to protein content of
cardiac myocytes assayed by the Lowry method.
Measurement of ATP in Cardiac Myocytes
The ATP content of myocytes was measured before hypoxia,
after 3 or 5.5 hours of hypoxia, and after 1 hour of
reoxygenation (Figure 1). Cardiac myocytes
(2.7x106 cells per dish) were treated with 0.25
mL of 0.6N ice-cold perchloric acid and centrifuged at
1000g for 5 minutes at 4°C. The supernatant was
neutralized with KOH to pH 5.0 to 7.0 and, after 10 minutes, was
centrifuged at 8000g for 5 minutes at 4°C to
remove the KClO4. The supernatant was used for
the assays. ATP was measured by high-performance liquid
chromatography (LC-9A liquid chromatograph,
Shimadzu) with a column of STR ODS-M
(Shimadzu).16
Statistical Analysis
Data are expressed as mean±SEM of 6 samples derived from 
6
separate experiments. Differences were analyzed by 2-way ANOVA
combined with Scheffé's test, and a P value of <0.05
was considered to be statistically significant.
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Results |
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Effects of Cilazaprilat, CV-11974, Angiotensin II, and Bradykinin on Hypoxia/Reoxygenation Injury
Treatment of myocytes with 5.5 hours of modified Tyrode solution followed by 1 hour of DMEM medium under normoxic conditions or hypoxia alone (5.5 hours) did not cause a significant release of CK into the myocyte culture media (data not shown). However, hypoxia followed by reoxygenation caused a significant increase in CK release, as shown in the control bar of Figure 2
. Figure 2 also shows the
effect of pretreatment with cilazaprilat, CV-11974,
angiotensin II, and bradykinin on this
reoxygenation-induced CK release. Cilazaprilat reduced
CK release in a dose-dependent fashion to 36% of control at
10-5 mol/L. Although CV-11974 is reported to
block the effect of angiotensin II and III, it did not
significantly inhibit CK release in the present study (Figure 2). In addition, there was no significantly additive increase in
CK release after administration of angiotensin II. In
contrast, bradykinin significantly lowered CK release in a
dose-dependent manner, with 10-6 mol/L
bradykinin reducing CK release to 37% of control.
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Cardioprotective Mechanism of Cilazaprilat Against
Hypoxia/Reoxygenation Injury
Reoxygenation-induced CK release in the control
and cilazaprilat-treated groups in the presence of Hoe 140,
staurosporine, L-NMMA, aminoguanidine,
indomethacin, and methylene blue is shown in Figure 3. Cilazaprilat
(10-5 mol/L) significantly reduced CK release
compared with control. However, the protective effect of cilazaprilat
was significantly inhibited by cotreatment with
10-6 mol/L Hoe 140 or
4x10-4 mol/L L-NMMA but not
2x10-9 mol/L staurosporine,
5x10-4 mol/L aminoguanidine, or
10-5 mol/L indomethacin.
Cotreatment with 10-5 mol/L methylene blue
partially blunted the cardioprotection by cilazaprilat.
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Bradykinin Accumulation in Myocytes and Nonmyocytes
During Hypoxia
Figure 4 show the time course of
bradykinin concentration in the culture media and
immunofluorescence stainings of myocytes,
nonmyocytes, and RAECs. Hypoxia significantly increased
bradykinin levels in the myocytes. Treatment of myocytes with
10-5 mol/L cilazaprilat significantly increased
bradykinin production to 4.5 times that of control after 5.5
hours of hypoxia. In contrast, hypoxia did not enhance
bradykinin production even in the presence of
10-5 mol/L cilazaprilat in nonmyocytes.
Hypoxia also significantly increased bradykinin levels in
RAECs, and 10-5 mol/L cilazaprilat significantly
enhanced bradykinin levels to 2 times that of control after 5.5 hours
of hypoxia.
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P<0.001 vs control.
cGMP Content in Cardiac Myocytes During Hypoxia
The time course of cGMP content change in the myocytes is
illustrated in Figure 5. cGMP content in
control cells did not change appreciably during the 5.5 hours of
hypoxia. However, cilazaprilat 10-5
mol/L markedly increased cGMP content with increased time of
hypoxia. This augmentation of cGMP production by
cilazaprilat was blunted by cotreatment with
4x10-4 mol/L L-NMMA or
10-5 mol/L methylene blue. In contrast,
5x10-4 mol/L aminoguanidine did not block the
cilazaprilat-induced increase in cGMP content. Bradykinin
(10-6 mol/L) significantly promoted cGMP
production throughout the hypoxic period.
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ATP Content in Cardiac Myocytes During
Hypoxia/Reoxygenation
The time course of ATP content change in myocytes is shown
in Figure 6. Exposure to 6.5 hours of
normoxic culture conditions alone did not affect ATP content in the
myocytes. In the control, hypoxia significantly lowered ATP
content in a time-dependent manner, and reoxygenation
induced a further decrease in ATP. Both 10-5
mol/L cilazaprilat and 10-6 mol/L bradykinin
significantly inhibited this
hypoxia/reoxygenation-induced decline in ATP.
The ATP content in the myocytes treated with cilazaprilat was 1.31
times that of control after 5.5 hours' hypoxia and 4.84 times
that of control after 1 hour of reoxygenation. L-NMMA
4x10-4 mol/L totally inhibited this
cilazaprilat-induced preservation of ATP.
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Discussion |
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In the present study, we have demonstrated that an ACE inhibitor, cilazaprilat, shows remarkable protection against hypoxia/reoxygenation injury in a dose-dependent fashion. Recent reports demonstrated the existence of a local renin-angiotensin system within the myocardium4 and indicated that angiotensin II type 1 receptor antagonist improves ischemia-reperfusion injury.17 In the present study, however, pretreatment with the angiotensin II receptor antagonist CV-11974 or angiotensin II did not worsen CK release (Figure 2
),
which strongly suggests that cilazaprilat reduced myocardial
hypoxia/reoxygenation injury independently of
angiotensin II synthesis.
Our results also show that the cardioprotective effect of cilazaprilat
is blocked by Hoe 140, which suggests that its effect is mediated by
the bradykinin B2 receptor (Figure 3).
Furthermore, we demonstrated that bradykinin production during
hypoxia was significantly increased by cilazaprilat (Figure 4). It has been reported that a local kinin-kallikrein system
exists in the heart18 and that myocardial bradykinin
levels are further enhanced by ischemia or ACE
inhibitors through their inhibitory effect on
the degradation of kinins.19
It is still unclear which cells produce the bradykinin in the heart.
One recent report20 suggested that the coronary
vascular endothelium is the main source of the release
of kinins. However, it is likely that myocytes also play a significant
role because bradykinin levels were significantly enhanced by
cilazaprilat after hypoxia in myocytes but not in
nonmyocytes under our culture conditions (Figure 4). In
addition, we have confirmed that nonmyocytes consist of 95%
fibroblasts and 1% to 2% endothelial cells.
Furthermore, the enhancement in bradykinin production by
cilazaprilat in myocytes was greater than that in RAECs. Although these
results were obtained from in vitro studies using neonatal cardiac
myocytes, the present data strongly suggest that myocytes may be an
important source of bradykinin and that ACE inhibitors may
act directly on myocytes through a local kinin-kallikrein system,
thereby contributing to cardioprotection in an autocrine/paracrine
fashion. Indeed, recent data indicate that kallikrein activity can be
detected in primary cultures of neonatal rat cardiocytes and in
heart slices.18
Previous observations have also demonstrated the existence of
functional bradykinin B2 receptors on
cardiomyocytes, which are coupled to the activation of
phospholipase C, the subsequent generation of inositol
1,4,5-triphosphate or diacylglycerol, an increase in cytosolic
Ca2+ levels, and the activation of
PKC.21 22 23 Elevated cytosolic Ca2+
can stimulate phospholipase A2 and
cyclooxygenase, as indicated by the
production of prostaglandins.24
Furthermore, the increase in cytosolic Ca2+
through the B2 receptor may also stimulate
myocyte constitutive NO synthase (cNOS).25 26 In the
present study, the cardioprotective effect of cilazaprilat was
significantly blocked by L-NMMA but not by staurosporine,
aminoguanidine, or indomethacin (Figure 3),
therefore suggesting that the effect of cilazaprilat is not mediated by
PKC, inducible NOS, or prostaglandins but rather by a
cNOS-associated, bradykinin B2 receptor–mediated
pathway.
The present study also indicates that the protective effect
of cilazaprilat is mediated by cGMP, because methylene blue
significantly blocked this effect (Figure 3). In addition, cGMP
content was significantly increased in hypoxic myocytes after treatment
with cilazaprilat, and this augmentation of cGMP was blunted by
cotreatment with L-NMMA and methylene blue but not aminoguanidine
(Figure 5). The data therefore suggest that cGMP
production in myocytes treated with cilazaprilat is mediated by
a cNOS-NO–guanylate cyclase signaling pathway. It has been
reported previously that cGMP can improve the energy state in the
ischemic heart.27 Indeed, in the present
study, cilazaprilat as well as bradykinin significantly preserved the
ATP content of myocytes (Figure 6).
Although the role of cGMP in regulating myocardial contraction remains controversial, recent reports suggest that cGMP can regulate sarcolemmal Ca2+ influx through L-type Ca2+ channels (ICa) by activation of a cGMP-stimulated phosphodiesterase28 29 or by cGMP-dependent protein kinase (PKG)30 31 and can reduce the myofilament response to Ca2+ via activation of an endogenous cGMP-dependent protein kinase (cGMP-PK).32 Furthermore, cGMP has been recently reported to mediate the negative inotropic effect of NO.33 34 It is therefore tempting to speculate that NO production induced by cilazaprilat modulated myocyte contractility and contributed to the energy-sparing effect, although we cannot exclude the possibility that other effects of NO, such as radical scavenge action, also contribute to cardioprotection.35
In conclusion, the present study demonstrates that cilazaprilat can protect isolated myocytes against hypoxia/reoxygenation injury, possibly as a result of bradykinin accumulation and the resultant production of NO by cNOS in hypoxic myocytes in an autocrine/paracrine fashion. NO may increase cGMP synthesis in myocytes, which may consequently modulate their contractility and may contribute to energy preservation and cardioprotection.
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Acknowledgments |
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The authors are grateful to Dr Henry Fliss, Department of Cellular and Molecular Medicine, University of Ottawa, Canada, for the generous academic advice, and to Dr Shinji Fushiki, Department of Dynamic Pathology, Kyoto Prefectural University of Medicine, for the technical advice of immunohistochemical examination.
Received July 2, 1998; revision received September 23, 1998; accepted October 5, 1998.
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