Poster
# 52
Poster |
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6th Internet World Congress for Biomedical Sciences |
Toshihiro Yorozuya(1), Naoto Adachi(2), Masao Soutani(3), Kazuo Nakanishi(4), Kentaro Dote(5), Shigeo Kimura(6), Takumi Nagaro(7), Tatsuru Arai(8)
(1)(2)(3)(7)(8)Ehime-university School of Medicine - Japan
(4)(5)(6)Ehime university School of Medicine - Japan
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[Pharmacology] |
[Cardiolovascular Diseases] |
There are many studies both morphological and functional, that glucocorticoids improve ischemia-induced myocardial injury (1-4). This action is speculated to be caused by the preservation of cellular functions in ischemia, which results in prolongation of the period of myocardial viability. Although glucocorticoids have been demonstrated to improve lactate imbalance and prevent leakage of intracellular enzymes caused by ischemia (3,4), the mechanism underlying this protection has not been clarified. Considering the improvement of lactate balance during continuing coronary occlusion, it is likely that glucocorticoids change metabolism in the anaerobic state. Since adenosine 5-triphosphate (ATP) provides energy for contraction and maintains membrane functional integrity, we investigated the effect of dexamethasone, a pure glucocorticoid, on the hypoxic reduction of ATP in the mouse heart.
This study was approved by the Committee on Animal Experimentation at Ehime University School of Medicine, Ehime, Japan. Male ddY mice each weighing about 40 g (Japan SLC, Shizuoka, Japan) were housed in groups in a room controlled at 23}1 degrees C and maintained in an alternating 12-h light/12-h dark cycle (lights on at 0600 h). Animals were deprived of food at least 6 h before the start of experiments because of the influence of the plasma concentration of glucose. Forty-eight mice were prepared and then assigned to four saline groups and four dexamethasone groups (6 animals in each). Saline or dexamethasone (5 mg/kg) was administered intraperitoneally. Three hours after the administration, mice were killed by decapitation, and the heart was immediately removed and incubated in a glucose-free hypoxic phosphate buffer equilibrated with N2 (pH 7.4, 37 degrees C). The heart was frozen in liquid nitrogen after 5, 10, or 20 minutes of incubation. In the non-hypoxic (0 min) group, the heart was frozen immediately after its removal. The temperature of the temporal muscle was measured at the time of decapitation. The frozen heart was weighed and quickly homogenized with ice-cold 3 mL of 0.4 mol/L perchloric acid. After centrifugation at 20,000~g for 30 minutes, the supernatant was injected into a high-performance liquid chromatography (HPLC) system to determine the tissue concentrations of ATP, adenosine 5-diphosphate (ADP), and adenosine 5-monophosphate (AMP). The HPLC system consisted of a pump (L-7100, Hitachi, Tokyo, Japan) used to deliver the mobile-phase, a model L-7250 sample injector (Hitachi) with a 100-L sampling loop, a separation column (GL-W510-S, 7.8~300 mm inside diameter, Hitachi), and a UV detector (L-7400, Hitachi). The mobile phase was 0.2 mol/L NaH2PO4, with an adjusted pH of 3.5 with 0.2 mol/L H3PO4, and the flow rate was 0.5 mL/min. The absorption intensity (peak height) was measured at a wavelength of 270 nm. For comparison of values in the dexamethasone group to those in the saline group at corresponding times, an unpaired t test was used.
The temperature of the temporal muscle measured after decapitation in the saline and dexamethasone groups was 37.8 0.4 and 37.6 0.4 degree C (mean +/- SD, n=6), respectively. No difference was observed between the dexamethasone and saline groups.
With respect to the ATP concentration, there were no differences between the two groups when the hearts were frozen immediately after removal (Fig. 1). The values were 1568 +/- 470 and 1727 +/- 259 nmol/g (mean +/- SD), respectively. Hypoxia for 5 minutes decreased ATP content to 330 +/- 130 nmol/g in the saline group. However, the extent of the ATP decrease caused by 5 minutes of hypoxia was significantly suppressed in the dexamethasone group. The ATP level was 1085 +/- 296 nmol/g. Although the long duration of hypoxia further lowered the ATP content in each group, no differences were found between the two groups after 10 or 20 minutes of hypoxia.
Similar to the decrease in ATP levels, heart ADP content decreased during hypoxia. Pretreatment with dexamethasone suppressed the extent of ADP decrease and, after 5 minutes of hypoxia, the effect was significant compared to the saline group (Fig. 2). In the dexamethasone group, the AMP content at 5 minutes of hypoxia was significantly low compared with that in the saline group (Fig. 3).
In the present study, dexamethasone attenuated a hypoxic decrease in the heart ATP concentration. This may be caused by an enhancement of ATP synthesis in the heart or a suppression of ATP consumption in an anaerobic state due to a decrease in the energy requirement. Since the ATP level at 0 minute did not differ between the groups, an increase in the ATP synthesis by dexamethasone is unlikely. Considering its beneficial effect on ischemic cardiac injury, it is conceivable that the agent reduces ATP consumption. In a canine study on myocardial ischemia, a pretreatment with methylprednisolone improved the segmental myocardial function and lactate imbalance, indicating the improvement of energy metabolism in an anaerobic state (4). These findings, taken together with the present result, indicate that glucocorticoids contribute to the improvement of ischemic damage by improving local energy metabolism. Glucocorticoids have been shown to increase the plasma glucose level, which may influence energy metabolism. In the current study, the effect of dexamethasone was evaluated by an isolated heart model, using a glucose-free medium. Therefore, it is unlikely that the glucose level affected the current result, although pretreatment with dexamethasone may have affected the intracellular level of glucose prior to hypoxia. Another element specific to glucocorticoids, besides hyperglycemia, seems to have provided the effect to preserve energy. There are some controversial reports that glucocorticoids do not provide protective roles in ischemia-induced change (5,6). However, there are differences in the methodology of positive and negative reports such as timing of drug administration, dosage, duration of ischemia, and animal species. Glucocorticoids were administered after ischemic events in most studies that had no beneficial effect. Since glucocorticoids seem to exert an influence on energy metabolism during hypoxia as shown in the current study, post-ischemic treatments may not contribute to the improvement of energy states.
Although, it might be difficult to correlate the present findings to the histologic outcome, the reduction in hypoxia-induced ATP consumption may be a mechanism responsible for the improvement of ischemic myocardial damage.
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[Pharmacology] |
[Cardiolovascular Diseases] |
