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Vol. 289, Issue 1, 93-102, April 1999
Department of Laboratory Medicine,
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Abstract |
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The site of action of 3-(2,2,2-trimethylhydrazinium) propionate (THP), a new cardioprotective agent, was investigated in mice and rats. I.p. administration of THP decreased the concentrations of free carnitine and long-chain acylcarnitine in heart tissue. In isolated myocytes, THP inhibited free carnitine transport with a Ki of 1340 µM, which is considerably higher than the observed serum concentration of THP. The major cause of the decreased free carnitine concentration in heart was found to be the decreased serum concentration of free carnitine that resulted from the increased renal clearance of carnitine by THP. The estimated Ki of THP for inhibiting the reabsorption of free carnitine in kidneys was 52.2 µM, which is consistent with the serum THP concentration range. No inhibition of THP on the carnitine palmitoyltransferase activity in isolated mitochondrial fractions was observed. These results indicate that the principal site of action of THP as a cardioprotective agent is the carnitine transport carrier in the kidney, but not the carrier in the heart.
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Introduction |
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It
is well known that certain fatty acids and their metabolites cause
injury to cardiac muscle cells (Corr et al., 1984
). In particular,
long-chain acylcarnitine inhibits the
Na+-K+ ATPase of sarcolemma
and the Ca++ ATPase of the endoplasmic reticulum,
and diminishes the contractility of cardiac muscle (Adams et al., 1979
;
Dhalla et al., 1992
). Hence, the development of drugs for
ischemic cardiopathy that suppress the synthesis of long-chain
acylcarnitine is in progress (Lopaschuk et al., 1988
; Dhalla et al.,
1992
; Anderson et al., 1995
).
3-(2,2,2-trimethylhydrazinium)propionate (THP, MET-88) represents one
such drug, which was synthesized by Institute of Organic Synthesis
(Riga, Latvia). The cardioprotective effect of THP is well established
(Dhar et al., 1996
; Kirimoto et al., 1996
; Aoyagi et al., 1997
; Akahira
et al., 1997
), and it is in clinical use in Latvia and Russia
(Sahartova et al., 1993
). In examining the major pharmacological
effect of THP, it is important to note that intramyocardial free
carnitine and long-chain acylcarnitine levels are decreased when THP is
administered to rats and guinea pigs (Simkhovich et al., 1988
; Dhar et
al., 1996
). However, the mechanism by which this reduction is
accomplished has not been clarified and, as a result, the site of
action of THP remains unknown.
THP is structurally similar to carnitine (Fig.
1). It is, therefore, possible that THP
inhibits the transport of free carnitine into the myocyte through the
cell membrane. Because free carnitine is a substrate of carnitine
palmitoyltransferase (CPT), it is also possible that THP competes with
free carnitine at the catalytic site of CPT in cardiac muscle.
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To test these hypotheses, the following experiments were performed: 1) THP was administered to mice, and free carnitine and long-chain acylcarnitine concentrations in the blood, heart, and urine were measured and the effect of THP on free carnitine transport to the heart and on free carnitine reabsorption by the kidney were analyzed, 2) myocytes and fibroblasts were prepared and the inhibitory effect of THP on free carnitine transport in these cells was examined, and 3) mitochondria were prepared from heart, and the inhibition of CPT-I and CPT-II activities by THP was examined.
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Materials and Methods |
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Reagents. THP was a gift from Taiho Pharmaceutical Company (Tokushima, Japan). Basal Medium Eagle (BME), Trypsin-EDTA and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY). Dulbecco's minimum essential medium obtained from Handai-Biken (Osaka, Japan). L-[3H]carnitine and [1-14C]acetyl-coenzyme A were obtained from Amersham (Amersham, UK). Unlabeled L-carnitine and RPMI-1640 were obtained from Sigma (Milwaukee, WI). Plastic dishes were obtained from Sumitomo (Tokyo, Japan). Triton X-100 and Clearsol I were obtained from Nakarai (Kyoto, Japan). The protein assay kit was purchased from Pierce (Rockford, IL). The Ultrafree-MC Centrifugal Filter Unit was purchased from Millipore (Bedford, MA). The carnitine assay kit was obtained from Kainos (Tokyo, Japan). A stomach tube was obtained from Natume Seisakusho (Osaka, Japan).
Animals. Age-matched (8-9 weeks old) male C57BL/6J mice (n = 283) weighing 24 to 29 g (Nihon Crea, Japan) were used throughout the study. One- to two-day old baby rats (n = 80) born from 10-week-old Sprague-Dawley rats and weighing 190 to 230 g from SLC (Hamamatsu, Japan) were only used for the preparation of myocytes. All procedures were performed according to the Guide for the Care and Use of Laboratory Animals, and were monitored by the Institutional Animal Care and Use Committee of the University of Tokushima.
Administration of THP.
THP was dissolved in water, and
0.5-ml aliquots were orally administered via a stomach tube to each fed
mouse under ether anesthesia at 10:00 AM each day. The doses of THP
used were set at 0, 100, 200, and 300 mg/kg based on the reported
values (Simkhovich et al., 1988
; Dhar et al., 1996
; Kirimoto et al.,
1996
).
Collection of Blood and Urine, and Extraction of Heart. Before the administration of THP, and at 1, 2, 4, and 5 days after administration, 0.05 ml of blood was collected by repeated tail cutting at 9:00 to 10:00 AM. Serum was separated and used for the assay for free carnitine and acylcarnitine.
Heart was sampled before the administration of THP, and 1, 2, and 5 days after administration. The heart was removed after an injection of sodium pentobarbital (0.05 mg/g i.p.) and immediately frozen in liquid nitrogen and stored. Free carnitine and acylcarnitine in the heart were measured. Excreted urine was collected in the periods between before administration and 24 h after administration, between 24 and 48 h after administration, and between 96 and 120 h after administration. Each urine sample was collected using a special metabolic cage for mice. Urine was passed through the mesh and collected at the bottom. Both the mesh and the bottom were washed throughout with distilled water and the volume was adjusted to 50 ml. The concentrations of free carnitine and acylcarnitine in each sample were measured.Measurement of Carnitine.
Free carnitine was determined by
radioisotopic assay (McGarry and Foster, 1985
) or spectrophotometric
assay by an enzymatic cycling reaction using a carnitine assay kit
(Takahashi et al., 1994
). Acylcarnitine in serum and urine was
determined as free carnitine after pretreatment with alkali and
subsequent neutralization. Short-chain acylcarnitine in a perchloric
acid extract and long-chain acylcarnitine in the acid-insoluble
fraction from heart were also determined as free carnitine after the
same pretreatment.
Effect of THP on CPT Activity.
After an injection of sodium
pentobarbital (0.05 mg/g i.p.), the heart was removed and mitochondria
were prepared from this tissue by the method of McGarry et al. (1983)
.
The mitochondrial fraction obtained was dissolved in a solution
containing 5 mM Tris-Cl (pH 7.4) and 150 mM KCl (Sample I). Sample II
was prepared by treating Sample I with 0.37% Triton X-100 (Woeltje et
al., 1987
).
Measurement of Free Carnitine Transport Activity in Myocytes and
Fibroblasts.
Myocytes were prepared by the method of Kaneko and
Goshima (1982)
. In brief, the heart was excised from one- or two-day
old Sprague-Dawley rats, minced in BME, and washed twice with PBS (
).
The minced tissue was suspended in 0.125% trypsin-EDTA and incubated
at 37°C for 15 min. Desegregated cells in the upper layer were
collected at the end of three incubation periods.
Ultrafiltration. A 0.3 ml aliquot of collected serum was placed on an Ultrafree MC Centrifugal Filter Unit and centrifuged at 3000g for 15 min at 30°C. The carnitine concentration in the solution recovered in the lower chamber (B) and that before filtration (A) were measured by radioisotopic assay, and the unbound fraction (fp) in serum, B/A × 100 (%), was obtained.
Measurement of Blood THP Concentration.
On the days of the
first, second, and fifth administration, blood was collected from the
abdominal aorta at 1, 2, 3, 6, 12, and 24 h after administration
of THP after an injection of sodium pentobarbital (0.05 mg/g i.p.).
Blood was obtained from one mouse, one time. Serum was separated and
0.15 ml of serum from each of three animals was pooled. THP
concentration was measured using methods described by Sahartova et al.
(1993)
.
Kinetic Analysis of Free Carnitine Transport in Heart and
Kidney.
In the in vivo condition, free carnitine transport in
heart was analyzed kinetically by assuming a carrier-mediated transport for the uptake of free carnitine and a passive diffusion for the efflux
as shown in Fig. 2A. Competitive
inhibition was also assumed for THP based on our preliminary
experiments in fibroblasts (data not shown) and the following equation
was derived for the kinetic analysis of heart to serum concentration
ratio (Kp) of free carnitine by
assuming steady-state conditions.
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(1) |
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(2) |
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(3) |
Statistical Analysis.
Data were analyzed statistically by a
two-way ANOVA (dose and time) and a one-way ANOVA followed by posthoc
Fisher's Protected least Significant Difference with the level
of significance set at p < .05. Kinetic analyses were
performed by the nonlinear least square's method MULTI (Yamaoka et
al., 1981
), and kinetic parameters such as
Km and
Vmax values were estimated.
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Results |
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Effects of THP on Carnitine Concentration in Serum and Heart.
Serum levels of free carnitine were decreased 1 day after the
administration of THP at doses of 100, 200, and 300 mg/kg, respectively (Fig. 3). Five days after administration,
serum free carnitine concentrations were further decreased. Based on
the two-way ANOVA, no statistical difference with respect to the dose
of THP was observed. However, a significant difference was observed
with respect to time (p < .05). The
acylcarnitine/free carnitine ratio in serum was 0.57 ± 0.20 before administration of THP, and no statistical difference with
respect to either dose nor time was observed.
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Transport of Free Carnitine from Serum to Heart.
The
Kp of free carnitine was then
calculated based on the serum and heart concentration as shown in Fig.
5. This parameter reflects the extent of
tissue distribution of carnitine. The
Kp increased with decreasing levels of
serum free carnitine after the administration of THP, although THP
would be expected to decrease the free carnitine transport from serum
to heart.
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Effect of THP on the Renal Handling of Free Carnitine.
These
experimental results suggest that the long-chain acylcarnitine in
heart, which is generally thought to be a key compound in determining
the effect of THP, is not governed by the intracellular effect of THP,
but, rather, by extracellular interaction with THP. As shown in Fig.
7, a strong correlation exists between
serum free carnitine concentration and long-chain acylcarnitine
concentration in heart (p < .0023). This relationship
suggests that the alteration of serum free carnitine by THP may be a
determining factor for the variation of long-chain acylcarnitine in
heart.
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Discussion |
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It has been reported that THP decreases intramyocardial free
carnitine and long-chain acylcarnitine levels when administered to rats
and guinea pigs (Simkhovich et al., 1988
; Dhar et al., 1996
). We
administered THP to mice and found a similar decrease in heart tissue.
Although
-butyrobetaine, a carnitine-analog, is incorporated
into cells (Christiansen and Bremer, 1976
), THP, which is also a
carnitine-analog, appears to be incorporated into cardiac muscle cells.
As a result, it has been generally thought that the mechanism of
decreased long-chain acylcarnitine involves the possible competitive inhibition of CPT by THP at its catalytic site (Bremer, 1983
). The
effect of THP on CPT was examined at 500 and 1000 µM, because heart
free carnitine levels are 500 to 700 nmol/g in mouse heart (Kuwajima et
al., 1998
). The activities of CPT-I and -II were not inhibited by 60 or
3000 µM THP. Therefore, it seems unlikely that THP inhibits CPT
activity in the cytosol and acts in a protective manner on cardiac
muscle, even if THP is actually transported into the cells. The level
of long-chain acylcarnitine in heart is related to that of free
carnitine (Fig. 4). This suggests that the decreased level of
long-chain acylcarnitine is caused by a decrease in free carnitine
level in heart.
Regarding the mechanism by which free carnitine is decreased, it has
been reported that THP inhibits the activity of free carnitine
synthesizing enzyme,
-butyrobetaine hydroxylase, in the liver in a
noncompetitive manner (Simkhovich et al., 1988
). However, the carnitine
content in all body tissues, including heart, is determined by not only
synthesis in vivo, but also by important factors such as ingestion,
absorption, membrane transport, and renal excretion (Borum, 1983
).
Therefore, even if carnitine synthesis is reduced, the amount of
decrease due to this reduced synthesis could be canceled by dietary
ingestion and renal resorption, and, as a result, the content in the
cardiac muscle may not be substantially decreased.
As shown in Fig. 5, the Kp value
becomes higher with decreasing free carnitine concentrations in serum
after the administration of THP. The kinetic analysis of carnitine
transport in heart in vivo suggests a high affinity for carnitine
(Km = 4.0 µM) and low affinity for
THP (Ki = 1300 µM) as summarized in
Table 2. In cultured myocytes, we found evidence for the direct
inhibition of free carnitine transport by THP. The
Km value for free carnitine in
cultured myocytes was 93.2 µM, which is close to the reported value
of 60 µM (Bahl et al., 1981
). Free carnitine transport was inhibited
by THP with a Ki of 1340 µM, which
is quite similar to the estimated Ki
value in vivo conditions. Thus, the decreased concentration of free
carnitine in heart after the administration of THP cannot be explained
by the inhibition of free carnitine transport to cardiac muscle cells
by THP.
Free carnitine in blood is filtered through the renal glomeruli and
reabsorbed into renal proximal tubular cells by means of a carnitine
transport carrier, which is localized in the apical side of the tubular
cells (Stieger et al., 1995
). Free carnitine transport is
Na+-dependent and the
Km for free carnitine has been
reported to be 55 µM (Rebouche and Mack, 1984
) or 17 µM (Stieger et
al., 1995
), based on experiments using kidney brush-border membrane
vesicles. When this reabsorption system is functioning normally, the
blood level appears to be maintained at a constant value (Mancinelli et
al., 1995
). When THP was given to mice, blood carnitine levels decreased as shown in Fig. 3. Interestingly, this decrease was rapid,
reaching a level below 50% of the level observed at 24 h after
THP administration. As the cause of such a rapid decrease, the strong
inhibition of renal reabsorption of free carnitine by THP represents
the most likely initial event. Hence, the inhibition of renal
reabsorption of free carnitine by THP in vivo was examined.
In our present examination, the free carnitine reabsorption rate was
approximately 96% under normal conditions of free carnitine concentration at 31 µM. This value is in agreement with the
reabsorption rate (about 93% at 30 µM) obtained by experiments
involving the renal perfusion system of rats (Mancinelli et al., 1995
).
Based on the evidence of competitive inhibition of THP on free
carnitine transport in heart and fibroblasts, it is reasonable to
assume that the CLr increased with an increase of
THP serum concentration (Fig. 8). We have assumed that there is no
secretion of carnitine from serum to urine in this analysis. Recently,
it has been reported that free carnitine, derived from acylcarnitine, is secreted into urine to some extent in an isolated perfused rat
kidney (Mancinelli et al., 1995
; Evans et al., 1997
). If this is the
case, the estimated Ki for THP (52.2 µM) would be lower. The Ki of THP in
kidney is much smaller than the Km of
free carnitine, which is completely opposite to the case in heart. The
Km of free carnitine is much smaller
than the Ki of THP in heart. These
results demonstrate the heterogeneity of carnitine-transport carriers among organs.
The reason why THP lowers free carnitine transport activity appears to
lie with its structural similarity to free carnitine. As examples of
inhibition of free carnitine transport activity by carnitine analogs,
-butyrobetaine, acetylcarnitine, and lysine are known to function as
inhibitors (Huth and Shug, 1980
; Bahl et al., 1981
). However, only THP
is used as a drug.
Given the data collected herein, it is possible to discuss speculative
adverse reactions of THP. Considering the reactions, the analytical
results of Juvenile Visceral Steatosis mice, which are congenitally
deficient in free carnitine transport activity may be cited. This type
of mice was reported to have a systemic carnitine deficiency (Kuwajima
et al., 1991
). A defect in renal free carnitine reabsorption in this
strain and loss of the saturable uptake of free carnitine transport
activity in fibroblasts have also been reported (Horiuchi et al., 1994
;
Kuwajima et al., 1996
).
Juvenile Visceral Steatosis mice show cardiomegaly with age, and an
increased number of mitochondria, along with numerous myelin-like
structures in their myocytes, as evidenced by histological examination
(Miyagawa et al., 1995
; Kuwajima et al., 1998
). In addition, numerous
ragged red fibers and an increased number of mitochondria were found in
skeletal muscles by Gomori-trichrome staining (Kaido et al., 1997
). An
abnormal increase in mitochondria was found even in extrinsic muscle
and diaphragm (Narama et al., 1997
); in addition, a distinct fatty
liver with triacylglycerol accumulation was found (Kuwajima et al.,
1991
). Therefore, when THP is administered, particularly in a large
dose, attention should be paid to injuries to these organs, although
the decrease in carnitine levels caused by THP is reversible.
In summary, the principal mechanism for the decreased carnitine concentration in heart did not involve the inhibition of free carnitine transport and/or metabolism in heart, but rather the increased renal clearance of carnitine, possibly by the inhibition of reabsorption. These results also point to the heterogeneity in carnitine transport carriers among organs.
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Acknowledgments |
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We thank Pharmacokinetics Research Laboratory, Taiho Pharmaceutical Co., Ltd., for performing the measurements of THP levels in serum. We also are grateful to Ms. Shiho Nakai and Ms. Misa Bando for skillful secretarial help, and Kouichiro Taniguchi for expert technical assistance.
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Footnotes |
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Accepted for publication October 28, 1998.
Received for publication June 30, 1998.
1 This work was partly supported by grants-in-aid from the Ministry of Education, Science, and Culture of Japan.
Send reprint requests to: Dr. Masamichi Kuwajima, M.D., Ph.D., Department of Laboratory Medicine, School of Medicine, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima, 770-8503 Japan.
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Abbreviations |
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THP, 3-(2,2,2-trimethylhydrazinium)propionate; CPT, carnitine palmitoyltransferase; BME, Basal Medium Eagle; fp, unbound fraction of free carnitine; Kp, heart to serum concentration ratio; CTHP, concentration of THP in serum or medium; Ccar, concentration of free carnitine in serum or medium; GFR, glomerular filtration rate; CLr, renal clearance of free carnitine; FBS, fetal bovine serum.
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