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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
JPET 292:838–845, 2000
Vol. 292, No. 3
Printed in U.S.A.
Targeted Antioxidant Properties of N-[(Tetramethyl-3-pyrroline3-carboxamido)propyl]phthalimide and Its Nitroxide Metabolite
in Preventing Postischemic Myocardial Injury1
RAVI A. SHANKAR, KALMAN HIDEG, JAY L. ZWEIER, and PERIANNAN KUPPUSAMY
Department of Medicine, Division of Cardiology and the EPR Center, Johns Hopkins University, School of Medicine, Baltimore, Maryland; and
Institute of Organic and Medicinal Chemistry, University of Pécs, Pécs, Hungary (K.H.)
Accepted for publication November 19, 1999
This paper is available online at http://www.jpet.org
Reperfusion of ischemic myocardium is known to be associated with a variety of ventricular arrhythmias and myocardial dysfunction that can lead to severe cardiac impairment
and cell death (Manning et al., 1984; Pogwizd and Corr, 1986;
Forman et al., 1990; Yamada et al., 1990). Lipid peroxidation
of myocardial cell membranes by reactive oxygen species
(ROS) such as superoxide anion (O2. ), hydrogen peroxide
(H2O2), hydroxyl radical (䡠OH), and singlet oxygen (1O2) has
been implicated as a potential mechanism for these deleterious effects (Kako, 1987; Halliwell et al., 1992; Esterbauer et
al., 1993). Over the years, a variety of therapeutic approaches
to protect the myocardium against these oxidants have been
investigated. Although the formation of ROS is known to
occur after the reperfusion of ischemic organs (Zweier et al.,
1987, 1989), questions remain regarding the most effective
Received for publication June 30, 1999.
1
This work was supported in part by National Cancer Institute Grant
CA78886, National Institutes of Health Grant HL38324, Hungarian Research
Foundation OTKA Grant T 021277, and Hungarian Academy of Sciences
Grant AKP 97-13 4,2 (K.H.). P.K. was supported by an Established Investigator Award from the American Heart Association during the tenure of this
study.
reperfusion injury. Serial measurements of contractile function
were performed on hearts subjected to ischemia-reperfusion.
Hearts were either untreated or treated with 50 ␮M TPC-NH or
with its metabolites for 1 min before ischemia and during the
first 5 min of reflow. TPC-NH showed marked protection with a
more than 3-fold increased recovery of contractile function
compared with control hearts, whereas its oxidative metabolites exhibited significant but lower protection. Thus, TPC-NH
and, to a lesser extent, its oxidation metabolites exhibit potent
membrane-targeted antioxidant action and exert marked protection against myocardial injury in the postischemic heart.
therapeutic approach to prevent the detrimental effects of
this oxidant injury. Prior studies have evaluated the efficacy
of antioxidative enzymes such as superoxide dismutase or
catalase or specific inhibitors of ROS-generating enzymes
such as allopurinol or oxypurinol (Jolly et al., 1984; Manning
et al., 1984; Pryzyklenk and Kloner, 1986). However, variable
protection was observed with these enzymes, and this may be
due to their inability to access intracellular region or limited
efficacy against only one type of oxidant. Thus, there has
been a need to develop antioxidant drugs that are both
readily internalized and able to scavenge a range of ROS
(Black et al., 1994; Kilgore et al., 1994).
Small-molecular-weight, stable nitroxides have been
shown to have potential therapeutic values in a variety of
disease processes, including myocardial reperfusion injury
(Gelvan et al., 1991; Samuni et al., 1991; Mohsen et al., 1995;
Zhang et al., 1998a), trauma (Zhang et al., 1998b), ulcerative
colitis and mucosal injury (Karmeli et al., 1995), radioprotection (Hahn et al., 1992), leukocyte-endothelial cell adhesion
(Russel et al., 1998), and doxorubicin (Adriamycin)-induced
cardiotoxicity (Monti et al., 1996). The protective effects of
ABBREVIATIONS: ROS, reactive oxygen species; TPC-NH, N-[(2,2,5,5-tetramethyl-3-pyrroline-3-carboxamido)propyl]phthalimide; TPC-NOH,
N-[(1-hydroxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-carboxamido)propyl]phthalimide; TPC-NO, N-[(1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-carboxamido)propyl]phthalimide; AAPH, 2,2⬘-azobis-2-amidinopropane dihydrochloride; DTPA, diethylenetriaminepentaacetate; X, xanthine; XO, xanthine oxidase; EPR, electron paramagnetic resonance; LVEDP, left ventricular end-diastolic pressure; LVSP, left ventricular systolic pressure;
LVDP, left ventricular developed pressure; HR, heart rate; CF, coronary flow; RPP, rate-pressure product; LDH, lactate dehydrogenase.
838
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ABSTRACT
We investigated the cardioprotective efficacy of a new compound based on 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide (TPC-NH). Biochemical studies using electron paramagnetic resonance (EPR) spectroscopy suggest that TPC-NH is a
scavenger of reactive oxygen species. In vitro cellular studies
show that TPC-NH protects isolated cardiomyocytes against
oxidative damage caused by superoxide radicals. Ex vivo EPR
studies on the isolated rat heart indicate that the TPC-NH is
metabolically oxidized to the nitroxide form. Studies were also
performed in the isolated rat heart model to measure the efficacy of TPC-NH and its metabolites in preventing postischemic
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Targeted Antioxidant Properties of a Nitroxide Precursor
Materials and Methods
Chemicals
The drug TPC-NH (amine) and its oxidation metabolites TPCNOH (hydroxylamine) and TPC-NO (nitroxide) were synthesized as
previously reported (Hankovszky et al., 1986). The components of the
modified Krebs’ buffer solution, lactate dehydrogenase (LDH) diagnostic kit, lidocaine, xanthine (X), and xanthine oxidase (XO) were
purchased from Sigma Chemical Co. (St. Louis, MO).
Isolated Heart Perfusion
Female Sprague-Dawley retired-breeder rats (weight, 300 ⫾ 30 g;
Harlan Company, Boston, MA) were used. All experiments were
carefully conducted in compliance with the National Institutes of
Health guidelines for the use of laboratory animals. After complete
anesthesia (65 mg/kg pentobarbital i.p.), the heart was excised and
the ascending aorta was rapidly cannulated. Retrograde perfusion
was initiated according to the method of Langendorf at a constant
pressure of 80 mm Hg using modified Krebs-bicarbonate-buffered
perfusate containing 17 mM glucose, 120 mM NaCl, 25 mM
NaHCO3, 5.9 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, and 0.5 mM
EDTA. All perfusate solutions were routinely filtered through two
1.2-␮m Millipore filters and bubbled with 95% O2/5% CO2 gas mixture at 37°C. A side arm in the perfusion line located just proximal
to the aortic cannula allowed infusion of the drug solutions. The
drugs were infused at a dilution of 1:20, with respect to the coronary
flow (CF), using a Harvard Apparatus infusion pump. Contractile
functions of the heart were measured using a fluid-filled latex balloon inserted into the left ventricular cavity, through the atrioventricular valve. The balloon was connected via a hydraulic line to a
Spectramed P23XL pressure transducer with pressures amplified to
a Gould four-channel strip chart recorder as well as to a personal
computer equipped with MacLab data acquisition software. The balloon volume was adjusted to achieve an initial left ventricular enddiastolic pressure (LVEDP) of 10 ⫾ 2 mm Hg, and all subsequent
measurements were performed at the same balloon volume. The CF
was measured with a T106 Transonic small animal flowmeter just
proximal to the aortic cannulation.
Studies were performed in four groups: 1) control, 2) TPC-NH, 3)
TPC-NOH, and 4) TPC-NO, wherein the drug or saline control was
infused for 1 min before ischemia and during the initial 5 min of
reperfusion. Studies were also performed on two additional groups
wherein the TPC-NH was infused: 5) only during preischemia, or 6)
only during the first 5 min of reperfusion. Studies were performed
with at least seven hearts per group.
Myocyte Preparation
Adult rat ventricular myocytes were isolated according to an enzymatic technique. Briefly, 2- to 4-month-old Sprague-Dawley rats
were anesthetized with pentobarbital. The hearts were quickly removed and retrogradely perfused with a low Ca2⫹-, collagenase-, and
protease-containing bicarbonate buffer at 37°C. The perfusion was
terminated when the heart tissue became soft. The ventricles were
cut off, and the cardiac myocytes were mechanically desegregated.
Myocytes were then rinsed in a bicarbonate solution and finally
resuspended in HEPES buffer containing 1.0 mM Ca2⫹. The myocytes were then used in cell cytotoxicity studies.
LDH Assay
Cell toxicity studies were performed on isolated ventricular myocytes. The release of LDH after cell membrane damage due to expo-
Fig. 1. Structure of TPC-NH and its oxidation
products: TPC-NOH and TPC-NO. In biological
tissues, TPC-NH is oxidized to TPC-NO, which
undergoes reversible one-electron reduction to
TPC-NOH. TPC-NO is a free radical and can be
measured directly using EPR spectroscopy.
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nitroxides have been attributed to antioxidative processes,
which include: 1) superoxide dismutase-mimicking activity
(Krishna et al., 1992, 1996a), 2) induction of catalase-like
activity in hemeproteins (Krishna et al., 1996b), and 3) radical scavenging. In addition, the nitroxides are cell permeable, making it possible to provide both intracellular and
extracellular protection against oxidative stress. Recent
studies demonstrated that nitroxides inhibit lipid peroxidation (Cighetti et al., 1997), protect against H2O2-induced
cytotoxicity in Chinese hamster cells (Mitchell et al., 1991)
and cultured cardiomyocytes (Samuni et al., 1991; Mohsen et
al., 1995), and prevent postischemic reperfusion injury in the
isolated heart (Gelvan et al., 1991). In biological tissues, the
nitroxides are reduced to hydroxylamine form, and it has
been well established that these two forms of nitroxide coexist in tissues. The hydroxylamine has also been shown to
protect isolated cardiomyocytes against ROS-mediated injury, possibly due to a mechanism different from that of
nitroxides (Zhang et al., 1998b).
A group of pyrroline-based compounds (Fig. 1) have been
shown to possess class I antiarrhythmic activity (Hankovszky et al., 1986; Krishna et al., 1998; Xue et al., 1998). The
amino compound N-[(2,2,5,5-tetramethyl-3-pyrroline-3-carboxamido)propyl]phthalimide (TPC-NH) is oxidized to the
hydroxylamine (TPC-NOH) and nitroxide (TPC-NO) in mice
(Twomey et al., 1997). The TPC-NH and TPC-NOH are diamagnetic, whereas the TPC-NO is a stable paramagnetic
molecule and can be directly detected by electron paramagnetic resonance (EPR) spectroscopy. Also recently, it has
been reported that the TPC-NH suppresses various canine
ventricular arrhythmias (Xue et al., 1998). We provide direct
evidence for the protective effects of TPC-NH and its oxidation metabolites in an isolated rat heart model. We demonstrate that these compounds exhibit markedly enhanced protection against reperfusion injury, presumably due to a
combination of antioxidative and antiarrhythmic mechanisms.
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Shankar et al.
sure to X/XO was measured. The isolated myocytes were divided into
four groups: 1) control, 2) cells ⫹ TPC-NH, 3) cells ⫹ X/XO, and 4)
cells ⫹ X/XO ⫹ TPC-NH. Six experiments were performed in each
group. The samples were mixed with 170 ␮M NADH and 0.1 M
phosphate buffer (pH 7.5) containing 600 ␮M sodium pyruvate. The
mixture was immediately transferred to a 1-ml quartz cuvette, and
absorbance was recorded at 340 nm at 30-s intervals for 10 min using
a Hewlett-Packard 8452A diode-array spectrophotometer. The activity of LDH was calculated from the rate of NADH oxidation and
expressed as LDH U/ml of cell suspension.
Assessment of Cell Death
EPR Spectroscopy
In Vitro EPR Characterization of Oxidation Products
of TPC-NH. The effect of a variety of oxidants including superoxide
(O . ), hydrogen peroxide (H2O2), singlet oxygen (1O2), ferryl (Fe4⫹),
and alkylperoxide (ROO䡠) on TPC-NH was studied using EPR spectroscopy. X (0.5 mM) and XO (0.02 U/ml) in aerobic phosphate buffer
at pH 7.4 containing 0.1 mM diethylenetriaminepentaacetic acid
(DTPA) were used to generate O2. radicals. Catalase (500 U/ml) was
included to scavenge hydrogen peroxide in the solution. Singlet oxygen was generated by the photoexcitation of rose bengal (Kukreja et
al., 1991). The ferryl species were generated using horse myoglobin
and hydrogen peroxide (Krishna et al., 1996b). 2,2⬘-Azobis-2-amidinopropane dihydrochloride (AAPH; 25 mM) was used in aerobic
solutions at 37°C to generate ROO䡠 radicals (Niki, 1990). EPR measurements were carried out using X-band (9.78 GHz) with a TM110
flat cell.
Measurements of TPC-NO in Whole Heart. EPR spectroscopy
measurements on the whole intact heart were performed using an
L-band EPR spectrometer with a reentrant resonator as described
previously (Kuppusamy et al., 1995). After infusion of the drugs, the
hearts were subjected to no-flow global ischemia. The hearts were
quickly removed from the perfusion setup, washed, and placed inside
the EPR sample cavity. Serial EPR spectra were acquired to continuously monitor the concentration of nitroxide (TPC-NO) in the heart
for the next 30 min of ischemic duration. The heart was maintained
at 37°C with a gentle flow of humidified warm air around the heart.
EPR Spectroscopic Measurement of TPC-NO from Heart
Effluents. After the infusion of drugs, the coronary effluents were
collected in 20-s aliquots during the preischemic and reperfusion
periods. Samples were immediately frozen in liquid nitrogen and
stored at 77 K until EPR measurements. The samples were thawed
and EPR spectra were recorded at room temperature using a flat cell
with IBM-Bruker ER 300 spectrometer operating at X-band with a
TM110 cavity. The spectrometer settings were: modulation frequency
of 100 kHz, modulation amplitude of 0.5 G, microwave power of 20
mW, and microwave frequency of 9.78 GHz. Spectra were acquired
as a sum of 10 scans with 30 s/scan sweep time. Spectral acquisition,
analysis, and quantification were performed as described previously
(Kuppusamy et al., 1995).
Ischemia-Reperfusion Protocol
After an equilibrium period of 15 min to allow for functional
stabilization, baseline values of CF, LVEDP, left ventricular systolic
pressure (LVSP), and heart rate (HR) were measured. The hearts
were then subjected to a 1-min, preischemic, controlled infusion of
drug/saline, at 5% of the CF rate, to achieve a final concentration of
50 ␮M in the perfusate. Subsequently, the hearts were subjected to
30 min of global, no-flow ischemia, followed by 45 min of reperfusion.
During the first 5 min of reperfusion, the hearts were also subjected
to a controlled infusion of the drugs at the same concentration and
flow rate as in the preischemic state. After the first 5 min, reperfusion was continued with the Krebs’ buffer alone for the remaining 40
min. Serial hemodynamic measurements were performed during the
period of reperfusion. Coronary effluents were collected, in 1.5-ml
aliquots, during the preischemic infusion period. The coronary effluent was also collected, in 20-s intervals, for the first 2 min of reperfusion and after this for a total reperfusion period of 45 min. The
aliquots containing the effluent was immediately frozen in liquid
nitrogen to preserve the drug and its oxidative metabolites until EPR
measurements.
Statistical Analysis
Data are presented as mean ⫾ S.E. Comparisons between groups
were made with a one-way ANOVA designed for repeated measures.
A P value of ⬍.05 was considered statistically significant
Results
Oxygen Radical-Mediated Reactions. To delineate the
efficiency of TPC-NH in scavenging oxygen-derived species,
we used EPR spectroscopy to monitor the generation of TPCNO, which is an oxidized product of TPC-NH. A solution of
TPC-NH in aerated phosphate buffer (0.1 M, pH 7.4) containing 0.1 mM DTPA, 5 mM X, and 0.02 U/ml XO did not show
any EPR signal of TPC-NO, suggesting that superoxide has
no effect on this compound. Also, no significant effect was
observed with ferryl myoglobin (50 ␮M). The reaction of
alkylperoxyl radicals with TPC-NH was examined using an
alkylperoxyl radical-generating compound, AAPH (Polysciences, Warrington, PA). The AAPH at ambient temperatures decomposes to produce alkyl radicals, which under
aerobic conditions react with oxygen to produce alkyl peroxyl
radicals. A time-dependent oxidation of TPC-NH to TPC-NO
was observed in presence of 25 mM AAPH under aerobic
conditions. Figure 2 shows the EPR spectra obtained after 15
min of incubation. The data suggest that TPC-NH can scavenge alkylperoxyl radicals and potentially inhibit lipid peroxidation reactions.
In Vitro Measurement of Protection against Oxidative Cellular Injury. It has been reported that both hydroxylamine and nitroxide forms protect cardiomyocytes against
oxidative cellular injury (Zhang et al., 1998a). This was
shown by measuring LDH release from isolated cardiomyocytes exposed to a flux of superoxide radicals. To investigate
whether the TPC-NH is also efficacious in protecting cardiomyocytes against superoxide-mediated oxidative cellular injury, in vitro cellular studies were performed. Freshly isolated adult rat left ventricular cardiomyocytes were exposed
to a steady flux of superoxide (O2. ), generated enzymatically
by the aerobic X/XO reaction in the presence of catalase (500
U/ml) to scavenge hydrogen peroxide. The experiment used
1 ⫻ 106 myocytes/ml with 0.5 mM X and 16 mU of XO at pH
7.4. The cellular damage caused by the superoxide radicals
was estimated by measuring the leakage of cytoplasmic
LDH. Figure 3 shows the amount of LDH leakage from cardiomyocytes subjected to 30 min of oxidative stress in the
presence and absence of TPC-NH. Myocytes in the absence of
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Cell death of isolated myocytes exposed to superoxide radicals was
assessed using the Trypan blue staining technique: 0.2 ml of the cell
suspension, 0.3 ml of Hanks’ balanced salt solution, and 0.5 ml of
0.4% Trypan blue solution were mixed together and allowed to stand
for 15 min. A small amount of this mixture was then transferred to
hemocytometer chamber and viewed under a microscope. All the
myocytes in the central 1-mm square and four 1-mm corner squares
were counted for staining. The myocytes were divided into four
groups: control, exposure to TPC-NH alone, exposure to X/XO in the
presence of catalase (500 U/ml), and exposure to X/X ⫹ TPC-NH in
the presence of catalase. The number of viable cells in each group
was counted and compared with the control group.
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Targeted Antioxidant Properties of a Nitroxide Precursor
841
using a hemocytometer. The results were expressed as a
percentage of the number of viable cells/100 cell count. It was
observed that cells incubated with X/XO ⫹ TPC-NH showed
a survival rate of 68 ⫾ 10%, whereas cells treated with X/XO
alone showed a survival rate of only 32 ⫾ 6%. Cells treated
with TPC-NH alone did not show any significant effect on the
survival rate compared with untreated cells. Thus, a reduction of more than 50% in cell death was observed in the
presence of TPC-NH.
In Vivo Measurement of TPC-NH Metabolites in the
Intact Heart. Isolated rat hearts were subjected to control
perfusion, followed by infusion of 100 ␮M TPC-NH, TPCNOH, or TPC-NO for 1 min. The hearts were then subjected
to no-flow global ischemia and quickly transferred to the
L-band EPR resonator. The EPR signal of TPC-NO was monitored continuously for up to 30 min. Typical EPR spectra of
TPC-NO are shown in Fig. 4. At the end of 30 min of ischemia, hearts were homogenized and treated with 10 mM
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Fig. 2. X-band (9.78 GHz) EPR spectra of TPC-NO derived from alkylperoxyl radical-mediated oxidation of TPC-NH. EPR spectra of TPC-NO
were measured from 1 mM TPC-NH in PBS containing 0.1 mM DTPA
solution aerobically incubated at 37°C without (a) and with (b) the alkylperoxyl radical-generating system AAPH (25 mM) for 15 min. The
TPC-NO showed a triplet spectrum with 14N hyperfine coupling constant
of 16.05 G. Spectral acquisition parameters were modulation amplitude
of 0.5 G, microwave power of 10 mW, and acquisition time of 5 min. The
strong triplet signal suggests that TPC-NH is oxidized to TPC-NO by
alkylperoxyl (ROO䡠) radicals.
Fig. 3. Bar diagram depicting ROS-mediated cellular injury in adult
ventricular myocytes. Cellular injury was assessed by measuring the
amount of LDH released from myocytes subjected to oxidative stress for
30 min at room temperature by the addition of X/XO, in both the presence
and absence of TPC-NH. Data are expressed as mean ⫾ S.E. in LDH
U/ml.
TPC-NH released 236 ⫾ 5 U/ml LDH, whereas in the presence of TPC-NH, the release of LDH was 114 ⫾ 4 U/ml. This
shows that TPC-NH exerted more than 50% protection of
myocytes from oxidative damage caused by ROS.
Cardiomyocytes subjected to oxidative stress as described
were also assessed for cell death using the trypan blue dye
exclusion method. After preparation of the cell suspension in
Hanks’ balanced salt solution, 0.5 ml of 0.4% trypan blue
solution was added. Fifteen minutes later, the viable cells
were counted in both the presence and absence of TPC-NH
Fig. 4. L-band (1.2 GHz) in vivo EPR spectra of TPC-NO from the heart.
Rat hearts were infused with 100 ␮M solutions of TPC-NH, TPC-NOH, or
TPC-NO for 1 min and subjected to no-flow global ischemia. The EPR
spectra of TPC-NO from whole intact hearts were measured continuously
for 30 min. Typical spectra acquired at 10 min of ischemia from hearts
infused with TPC-NH (a), TPC-NOH (b), TPC-NO (c) are shown. At the
end of 30-min ischemia, hearts were homogenized, and the homogenates
were treated with 10 mM ferricyanide to convert TPC-NOH to TPC-NO.
Right, spectra were obtained from the homogenates of hearts treated with
TPC-NH (d), TPC-NOH (e), or TPC-NO (f). The TPC-NO showed a threeline spectrum with 14N hyperfine splitting 15.5 and 16.4 G. Spectral
acquisition parameters were frequency of 1.2 GHz, modulation amplitude
of 1.0 G, and microwave power of 5 mW.
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Shankar et al.
Fig. 5. Recovery of contractile functions in isolated hearts subjected to
ischemia and reperfusion. Perfused rat hearts were subjected to 30 min of
global no-flow ischemia, followed by 45-min reperfusion. Hearts were
infused with 50 ␮M TPC-NH, TPC-NOH, TPC-NO, or buffer during 1 min
before ischemia and during the first 5 min of reperfusion. During reperfusion, LVDP, RPP (an index of mechanical function), and CF were
measured and expressed as the percentage of the corresponding preischemic baseline values. E, hearts perfused with only Krebs-bicarbonate
buffer (n ⫽ 12); F, hearts infused with TPC-NH (n ⫽ 7); f, hearts infused
with TPC-NOH (n ⫽ 7); Œ, hearts infused with TPC-NO (n ⫽ 7). Data are
plotted as mean ⫾ S.E.
reperfusion in hearts, which were infused with the TPC-NH
for 1 min before ischemia, showed enhanced recovery (P ⬍
.01) compared with the hearts that received the drug only
during reperfusion (Fig. 6).
The parent compound TPC-NH has been reported to have
antiarrhythmic properties (Hankovszky et al., 1986; Xue et
al., 1998). To evaluate the nature of protection, we compared
the protection offered by this compound with that of another
class I antiarrhythmic drug, lidocaine (Das and Misra, 1992).
As seen in Fig. 6, hearts treated with lidocaine showed significantly less recovery compared with hearts treated with
TPC-NH, suggesting that the protection observed in this
model is possibly augmented by its antioxidant property.
EPR Spectroscopy of Heart Effluents. Because it was
observed that TPC-NH was capable of scavenging hydrogen
peroxide (in the presence of trace metals), singlet oxygen, and
alkylperoxyl radicals to form the nitroxide radical TPC-NO,
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ferricyanide to selectively oxidize TPC-NOH to TPC-NO, and
their EPR spectra were measured. The spectra of TPC-NO
from hearts treated with TPC-NH, TPC-NOH, and TPC-NO
are shown in Fig. 4, a– c, respectively, whereas the spectra
after treatment with ferricyanide are shown in Fig. 4, e– g. A
characteristic triplet spectrum with 14N hyperfine-splitting
constants of 15.5 and 16.4 G was observed. The unequal
coupling constants observed in the low-frequency (1.2 GHz)
EPR spectra are due to the breakdown of the high-field
approximation at 427 G used in this experiment. The spectra
of ferricyanide-treated heart tissues correspond to the sum of
TPC-NOH and TPC-NO at the end of 30 min of ischemia.
Hearts treated with TPC-NH did not show any nitroxide (Fig.
4a) or hydroxylamine (Fig. 4d) at the end of 30 min of ischemia. This may suggest that TPC-NH was not metabolized to
nitroxide. On the other hand, hearts treated with TPC-NOH
was observed to show a small amount of TPC-NO (Fig. 4b),
but at the end of 30 min of ischemia, the ischemic heart
consisted entirely of TPC-NOH (Fig. 4, b and e). Hearts
loaded with TPC-NO showed reduction in TPC-NO at least
partially to its hydroxylamine (Fig. 4, c and f). At the end of
30 min of ischemia, it was observed that the entire amount of
TPC-NO was reduced to TPC-NOH (Fig. 4e). The results thus
suggest that at the end of 30 min of ischemia, the TPC-NH
was not oxidized, whereas TPC-NOH and TPC-NO remained
in the tissue mostly in the reduced form TPC-NOH.
Heart Functional Data. To evaluate the cardioprotective
action of TPC-NH and its oxidation metabolites, isolated rat
hearts were perfused with a 50-␮M concentration of each
compound during 1 min before ischemia and during the first
5 min of reperfusion. LVSP, LVEDP, HR, and CF were measured preischemia and during 45 min of reperfusion. In all
the hearts, the preischemic LVEDP value was adjusted to
10 ⫾ 2 mm Hg. The other preischemic baseline values were
LVSP, 160 ⫾ 42 mm Hg; HR, 320 ⫾ 24 beats/min; and CF,
16 ⫾ 5 ml/min. The left ventricular developed pressure
(LVDP) was computed as the difference between LVSP and
LVEDP. The rate-pressure product (RPP) was obtained as a
product of HR and LVDP. The data obtained during reperfusion were expressed as a percentage of their corresponding
preischemic baseline values. Figure 5 shows the recovery of
LVDP, RPP, and CF as a function of reperfusion time. The
percentage recovery of LVDP in the hearts infused with
TPC-NOH or TPC-NO was significantly higher (P ⬍ .01)
than that of control. At the end of 45 min of reperfusion, the
LVDP of hearts treated with TPC-NOH and TPC-NO showed
30 ⫾ 4 and 21 ⫾ 1% of recovery, respectively, compared with
11 ⫾ 1% for control (Fig. 6). The recovery of LVDP in TPCNH-treated hearts was even more significant (40 ⫾ 3%; P ⬍
.001) than its oxidative metabolites. Also, similar recoveries
were observed with respect to RPP values. The RPP values at
the end of 45 min of reperfusion were 38 ⫾ 3, 31 ⫾ 3, and
22 ⫾ 2% for hearts treated with TPC-NH, TPC-NOH, and
TPC-NO, respectively, compared with 11 ⫾ 1% for control
hearts. The recovery of CF in treated hearts was not different
(P ⬎ .05) from that of control hearts (Fig. 5), suggesting that
the three compounds did not alter the CF.
To further evaluate whether the cardioprotective action of
TPC-NH occurs during ischemic or during reperfusion phase,
hearts were treated with TPC-NH, either 1 min before the
onset of ischemia (group 5) or during the first 5 min of
reperfusion (group 6). The LVDP, RPP, and CF at 45 min of
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2000
Targeted Antioxidant Properties of a Nitroxide Precursor
843
intensity showed a similar increase during the first 20 to
40 s, and it gradually decreased thereafter.
Discussion
the effluents from reperfused hearts were collected and examined using EPR spectroscopy. Heart effluents were collected for every 20 s during the 45 min of reperfusion and
immediately frozen in liquid nitrogen. The frozen samples
were later thawed, and their nitroxide content was measured
and quantified as described in Materials and Methods. Figure 7 shows the intensity of TPC-NO signal as a function of
reperfusion time. The signal intensity showed an increase
during the first 20 to 40 s, and it gradually decreased thereafter. Hearts were also perfused with 50 ␮M TPC-NOH,
effluents collected and analyzed (data not shown). The signal
Fig. 7. Intensity of the TPC-NO signal, measured by X-band EPR spectroscopy, from the coronary effluents of the heart. Rat hearts were subjected to 30 min of global no-flow ischemia and reperfused for 45 min. The
coronary effluents were collected for the first 15 min of reperfusion and
measured for the EPR signal of TPC-NO. F, from hearts treated with
TPC-NH for 1 min before the onset of ischemia; E, from hearts treated
with TPC-NH only during the first 5 min of reperfusion. Results are
expressed as mean ⫾ S.E. Hearts pretreated with TPC-NH showed a
significant increase in the TPC-NO signal intensity during the first 1 min
of reperfusion.
⬎ NOOH ⫹ L䡠 (LO䡠, LOO䡠) ¡ ⬎NOO
⫹ LH (LOH, LOOH)
(1)
Because of the equilibrium between nitroxide and hydroxylamine in tissues, their concentrations in tissues are continuously replenished. This interconversion enables them to
act in a catalytic mode, contrary to common antioxidants,
which operate in a stoichiometric mode.
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 3, 2017
Fig. 6. Recovery of contractile functions at 45 min of reperfusion in
isolated hearts subjected to 30 min of global ischemia. Hearts were
infused with 50 ␮M TPC-NH, TPC-NOH, TPC-NO, lidocaine, or Krebs’
buffer during 1 min before ischemia and during the first 5 min of reperfusion. TPC-NH was also administered either only during the preischemic infusion [TPC-NH(PRE)] or only during the 5-min reperfusion period [TPC-NH(POST)]. The contractile functions are expressed as a
percentage of the preischemic baseline. Data are plotted as mean ⫾ S.E.
The TPC-NH is a five-member sterically hindered pyrroline-based compound characterized by three structural
groups: an aromatic ring that anchors the drug into the
lipophilic alkyl chains of membrane phospholipids, an amino
group that ionizes at pH 8 to 9, and an interconnecting chain
that joins the aromatic ring and the amino group and also
has substituents that are capable of hydrogen bonding. In
biological tissues, TPC-NH is converted to TPC-NO, which in
turn is bioreduced to give the corresponding hydroxylamine
TPC-NOH. This interconversion gives the potential of tissues
treated with TPC-NH to have all three forms of the compound coexisting at any time during the treatment period.
The ability of nitroxyl radicals to inhibit in vitro lipid
peroxidation was recently reported (Cighetti et al., 1997).
Lipid peroxidation induced by Fenton’s reagent in liver microsomes and egg phosphotidylcholine was found to be inhibited by stable lipophilic steroidal nitroxide radical. The steroidal nitroxide was shown to function as a chain-breaking
antioxidant. It was further observed that the inhibition was
comparable to that of ␣-tocopherol, which is a lipid-soluble
antioxidant known to work in fatty areas such as the lipids,
suggesting that a good affinity for cell membranes increases
the lipid peroxidation inhibitory effect. Although the mechanism of inhibition of lipid peroxidation in cell membranes by
nitroxides is due to the termination of lipid peroxyl radical
cascade, their intracellular protection against oxidative damage has been ascribed to the ability of nitroxides to oxidize
the reduced metals, such as iron and copper. The oxidation of
reduced metal ions will preempt the Fenton reaction and
prevent the formation of secondary 䡠OH radicals. It is also
possible that nitroxides offer cellular protection through reaction with intracellular O2. and secondary radicals such as
R䡠, RO䡠, or ROO䡠, terminating the propagation of radical
chain reactions.
In most of the research aimed at investigation of the therapeutic potential (antioxidant, antiarrhythmic, and radiation
protection) of the nitroxides, the role of hydroxylamine form
has not been investigated in detail. The bioreduction product
of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, or Tempol
(hydroxylamine), also showed significant protection of cardiomyocytes against cell membrane damage induced by X/XO
(Zhang et al., 1998b). The antioxidative activity of the hydroxylamine was found to be greater than that of the nitroxide itself (Zhang et al., 1998a). The mechanism of protection
by the hydroxylamine form has been suggested to be due to
detoxification of lipid radicals involving a hydrogen atom
abstraction reaction leading to chain termination and production of nonradical species:
844
Shankar et al.
Vol. 292
Fig. 8. Schematic of the action of TPC-NH in
the prevention of oxidant injury. TPC-NH is
similar to vitamin E in scavenging reactive
oxygen intermediates to form a nitroxide
radical (TPC-NO). This radical can be reduced by ascorbic acid to the diamagnetic
N-hydroxyl compound (TPC-NOH). The
semidehydroascorbate radical formed in this
process is reduced by cellular thiols. The
TPC-NOH is a nontoxic intermediate, which
is sufficiently reactive to reduce/scavenge reactive oxygen intermediates by transferring
its hydrogen atom.
beginning reperfusion) hearts (P ⬍ .05) suggests that there is
both ischemia- and reperfusion-associated protection. The
possibility that the protection could be due to the ability of
TPC-NH to function as an antiarrhythmic agent was considered. Because the functional recovery of hearts treated with
lidocaine was not as prominent as that of hearts treated with
TPC-NH, it is clear that there are additional pathways that
have an important role in its marked myocardial protection.
It is seen from Fig. 6 that there is a marked decrease in the
CF in hearts treated with lidocaine, suggesting that lidocaine
is vasoactive. The vascular effect of lidocaine in rat cremaster
muscle preparations has been reported (Johns et al., 1985). A
biphasic response to increasing concentrations of lidocaine
was observed. Progressive constriction was noted with lidocaine concentrations from 1 to 1000 ␮g/ml, whereas vasodilation was observed for 10 mg/ml lidocaine. In our experiments, lidocaine was used at a concentration of about 100
␮g/ml, so the decrease in CF seen in our hearts treated with
lidocaine is consistent with prior literature.
The particular design of TPC-NH and its metabolites having a lipophilic aromatic end and a hydrophilic amino group
at the other end separated by alkyl chain imparts these
compounds with the ability to detoxify oxygen radicals in the
lipid-rich membrane as well as water-rich cytosolic areas.
These types of compounds have been shown to have much
higher solubility in the aqueous/lipid membrane interface
(Subczynski et al., 1998). This may result in enhanced membrane activity leading to blockage of cardiac sodium and
calcium channels, thus offering ischemic protection. In addition, TPC-NH may also protect Na⫹,K⫹-ATPase function of
cardiac myocytes against ischemia and reperfusion-induced
inactivation. Our results show that TPC-NH is capable of
providing membrane stabilization by localized site-targeted
detoxification of ROS that are generated during reperfusion.
Thus, it appears that the compound is capable of providing
both ischemic and postischemic myocardial protection
against reperfusion injury.
The TPC-NH is able to take up oxygen from the most
reactive oxygen radicals to form nitroxide. This is similar to
vitamin E in that it scavenges reactive oxygen intermediates
(oxy radicals and also nonradicals) to form a nitroxide radical
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 3, 2017
The EPR measurements (Fig. 4) of tissue TPC-NOH, and
TPC-NO from hearts with 1-min preischemic infusion of
TPC-NH, TPC-NOH, and TPC-NO suggest that at the end of
30 min ischemia, that is, at the beginning of reperfusion, the
tissue consisted only of TPC-NH in the case of TPC-NHinfused hearts or TPC-NOH in the case of hearts infused
with the other compounds. Reintroduction of oxygen during
the reperfusion phase causes oxidative conversion of
TPC-NH and TPC-NOH to TPC-NO, which undergoes subsequent reversible bioreduction to TPC-NOH, thus maintaining a steady-state concentration of all the three compounds
in the reperfused tissue. Furthermore, preadministration followed by 30 min of ischemic duration results in complete
internalization of TPC-NH and its metabolites in cells. Because all three forms coexist at any time in tissue and because their antioxidant action is catalytic in nature, a comprehensive “sweep” of toxic oxygen radicals can occur,
leading to a more enhanced protection compared with the
presence of any single compound. The in vivo administration
of TPC-NH provides three different antioxidants in a steadystate concentration in tissues.
The present investigation demonstrates that all the three
compounds, administered separately, are capable of protecting against ischemia-reperfusion injury in isolated rat
hearts. However, the addition of TPC-NH for 1 min before
ischemia is significantly more protective than the addition of
TPC-NH at the start of reperfusion. The observations that
the oxidative metabolites are not made during ischemia and
that TPC-NH added before ischemia may be still present
during reflow may suggest that much of the protective effect
of TPC-NH is not due to metabolism or to the oxidative
metabolites. The protective effect of TPC-NH against postischemic injury could be due to: 1) reduction in the severity of
ischemic damage incurred by the deprivation of oxygen (ischemic protection), 2) a direct antiarrhythmic activity providing protection against arrhythmia-induced damage (antiarrhythmic protection), or 3) scavenging of toxic oxidative
species that cause cardiac damage during reperfusion (antioxidant protection). The observation that hearts pretreated
(1 min, preischemic) with TPC-NH showed a better recovery
of contractile function compared with post-treated (5 min,
2000
(Fig. 8). This radical can be reduced by ascorbic acid to the
labile diamagnetic N-hydroxyl compound (TPC-NOH). The
semidehydroascorbate radical formed in this process is reduced by cellular thiols. The advantage of this process is that
the TPC-NOH is a nontoxic intermediate, which is sufficiently reactive to reduce/scavenge reactive oxygen intermediates by transferring its hydrogen.
In summary, the pyrroline-based antiarrhythmic nitroxide
precursor compound TPC-NH is metabolized in vivo to form
the corresponding hydroxylamine and nitroxide derivatives
and offers the potential of membrane-targeted antioxidant
action against myocardial postischemic reperfusion injury.
Acknowledgments
We thank Dr. Murali C. Krishna for helpful comments and Bruce
Ziman for his assistance in the preparation of myocytes.
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