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.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Structures of THP and carnitine. Left, THP; right, carnitine.

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.

	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.

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 (K_p) of free carnitine by assuming steady-state conditions.

(1)

where the V_max and K_m represent the maximum transport rate and Michaelis-Menten constant for free carnitine by carrier-mediated transport. K_i represents the inhibition constant by THP and k represents the efflux rate constant. C_THP and C_car represent the serum concentration of THP and free carnitine.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Kinetic models for the transport of free carnitine and its inhibition by THP. A, distribution to heart. Carrier-mediated transport and passive diffusion were assumed for influx and efflux of free carnitine in heart. Competitive inhibition by THP was assumed. B, reabsorption of free carnitine in kidney. Carrier-mediated transport was assumed for the reabsorption of free carnitine in the apical membrane of proximal tubular cells.

(2)

(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 K_m and V_max values were estimated.

	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.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Time courses for free carnitine or long-chain acylcarnitine in serum and heart after administration of THP. THP doses were 100 mg/kg b.wt. (-), 200 mg/kg b.wt. (-), or 300 mg/kg b.wt. (-). Each data point represents the mean ± S.E. (n = 12 at time zero, n = 4 at other times). *p < .05, **p < .01, ***p < .001 compared before and after administration of THP.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Relationship between free carnitine and long-chain acylcarnitine in heart. Time courses for free carnitine and long-chain acylcarnitine concentration in heart were plotted. Each data point represents the mean value of Fig. 3. The solid line represents the regression line (r = 0.809, p < .0029).

Transport of Free Carnitine from Serum to Heart. The K_p 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 K_p 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.

View larger version (8K): [in this window] [in a new window]   Fig. 5.   Relationship between Kp and serum free carnitine. Kp represents the heart to serum concentration ratio of free carnitine based on the data shown in Fig. 3. The solid curve represents the simulation curve using equation (1) fixing the CTHP at 90.2 µM.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Concentration-dependent uptake of free carnitine by myocytes. Open and closed circles represent the uptake of free carnitine by myocytes with or without THP (3 mM), respectively. Each data point represents the mean of four experiments. The uptake was measured after 2 h at the indicated concentration. The solid curves represent the fitting curve based on equation (2) using the parameters summarized in Table 2.

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.

View larger version (10K): [in this window] [in a new window]   Fig. 7.   Relationship between serum free carnitine and heart long-chain acylcarnitine. Each data point represents the mean value of Fig. 3. The solid line represents the regression line (r = 0.818, p < .0023).

View larger version (9K): [in this window] [in a new window]   Fig. 8.   Relationship between serum free carnitine concentration and renal clearance of free carnitine. The CLr was calculated from the excreted free carnitine in urine divided by the area under the curve for serum free carnitine concentration for 24 h before killing the animals. The solid line represents the regression line (r = 0.863, p < .001).

View larger version (10K): [in this window] [in a new window]   Fig. 9.   Relationship between CLr and serum THP. CLr increases with increasing serum THP concentration, which was averaged for 24 h before killing animals. The solid curve represents the simulation curve by equation (3) fixing the Ccar at 50 µM.

	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 K_p 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 (K_m = 4.0 µM) and low affinity for THP (K_i = 1300 µM) as summarized in Table 2. In cultured myocytes, we found evidence for the direct inhibition of free carnitine transport by THP. The K_m 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 K_i of 1340 µM, which is quite similar to the estimated K_i 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 K_m 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 CL_r 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 K_i for THP (52.2 µM) would be lower. The K_i of THP in kidney is much smaller than the K_m of free carnitine, which is completely opposite to the case in heart. The K_m of free carnitine is much smaller than the K_i 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.