Effects of exercise on platelet and aortic functions in aged rats.

AIM AND METHODS: To assess age- and exercise-related changes in platelet aggregation, we measured the magnitude of platelet aggregation with a four-channel aggregometer, plasma and aortic polyunsaturated fatty acids by gas chromatography and related prostanoids with a reagent kit in young and aged non-exercised and in aged exercised rats. RESULTS: Platelet aggregation in platelet-rich plasma induced by ADP (5 microm) in the primary wave increased with age. In the non-exercised groups, the basal levels of thromboxane B2 in platelet-rich plasma increased in aged rats compared with young rats. In aged exercised rats, the basal levels of 6-keto-prostaglandin F1alpha in platelet-rich plasma were stimulated and those of thromboxane B2 were depressed, compared with non-exercised aged rats. The plasma levels of eicosapentaenoic acid and docosahexaenoic acid increased with age. Only aortic eicosapentaenoic acid in the aged group increased by exercise. In the aged non-exercised and exercised groups, the aortic, but not the plasma, levels of eicosapentaenoic acid correlated inversely with the basal levels of thromboxane B2 in platelet-rich plasma (r = -0.53, P < 0.05) and associated negatively with the magnitudes of platelet aggregation induced by ADP (5 microm) (r = -0.47, P < 0.05). CONCLUSION: These findings suggest that exercise in aged rats increases aortic eicosapentaenoic acid concentrations, which in turn depress the basal levels of thromboxane, B2 in platelet-rich plasma to modulate platelet aggregation.

Chronic administration of docosahexaenoic acid improves the performance of radial arm maze task in aged rats.

1. In the present study, we investigated the effect of docosahexaenoic acid (DHA) on spatial memory related learning ability in aged (100 weeks) male Wistar rats. 2. Rats were fed a fish oil-deficient diet through three generations and were then randomly divided into two groups. Over 10 weeks, one group was per orally administered 300 mg/kg per day DHA dissolved in 5% gum Arabic solution and the other group was administered the vehicle alone. Five weeks after the start of the administration, rats were tested with the partially baited eight-arm radial maze to estimate two types of spatial memory related learning ability displayed by reference memory error and working memory error. 3. Chronic administration of DHA significantly decreased the number of reference memory errors and working memory errors. 4. The level of lipid peroxide (LPO) in the hippocampus tended to decrease with chronic DHA administration and demonstrated a positive correlation with the number of reference memory errors. 5. These results suggest that the accumulation of hippocampal LPO reduces spatial memory related learning ability in aged rats. Moreover, chronic administration of DHA was effective in decreasing the level of hippocampal LPO, then improving learning ability.

Nicorandil stimulates release of adenyl purines from porcine coronary artery.

1. To clarify the mechanism of the cardioprotective effect of nicorandil (2-nicotinamidoethyl-nitrate ester), the effects of nicorandil and nitric oxide (NO) donors on the release of ATP, ADP, AMP and adenosine from arterial segments and cultured endothelial cells of the porcine coronary artery were examined. 2. Nicorandil significantly increased the release of total adenyl purines from arterial segments and from cultured endothelial cells. 3. Cromakalim, an ATP-sensitive K+ channel opener, did not affect the release of total adenyl purines from coronary artery segments. 4. s-Nitroso-N-acetyl-D,L-penicillamine and isosorbide dinitrate, NO donors, significantly increased the release of total adenyl purines from coronary artery segments. 5. These results demonstrate that nicorandil stimulates ATP release from the coronary artery by acting not as an ATP-sensitive K+ channel opener, but as a nitrate, thus suggesting the cardioprotective properties of nicorandil.

Heart preservation in HTK solution: role of coronary vasculature in recovery of cardiac function.

BACKGROUND: Poor myocardial tolerance to prolonged cold ischemia remains a major concern in heart transplantation. In this study, we estimated superiority of Histidine-Tryptophan-Ketoglutarate (HTK) over University of Wisconsin (UW) as a cardiac preservation solution. METHODS: Isolated rat hearts were mounted on a Langendorff apparatus to estimate the baseline cardiac function. The hearts were arrested and stored at 4 degrees C in UW and HTK solution for 8 hours, and then reperfused. The aortic flow, coronary flow, cardiac output, rate pressure product, and left ventricular dp/dt in the HTK group recovered significantly more than the UW group. The values of myocardial total adenine nucleotides and the adenosine triphosphate to adenosine diphosphate ratio were higher in the HTK than in the UW group. We also examined coronary vascular responsiveness using left coronary arteries dissected from the rat hearts before flushing, before storage, after storage, and after reperfusion. RESULTS: The maximal relaxation response to acetylcholine was significantly higher in the HTK than in the UW group after reperfusion, although there were no significant differences at each stage before reperfusion. In addition, the endothelium-independent relaxation response to sodium nitroprusside in the HTK group was also well preserved after reperfusion. CONCLUSIONS: These results indicate that HTK is superior to UW solution for cardiac preservation. HTK protects coronary vasculature during preservation, which together with reperfusion might lead to improved functional cardiac recovery following preservation.

Shear stress enhances prostacyclin release from endocardial endothelial cells.

The effect of shear stress on the release of prostacyclin (PGI2) from cultured endocardial endothelial cells (EECs) was investigated. EECs were harvested from the right ventricle (RV) and the left ventricle (LV) of porcine heart. Confluent EECs were incubated under various degrees of shear stress (0.2, 1, 4 and 6 dyne/cm2) and PGI2 release from each cell was measured. PGI2 release from LV-EECs and RV-EECs was enhanced by the elevation of shear stress in a shear-dependent manner with a rapid increase at the onset of flow; however, there was no significant difference in PGI2 production between RV-EECs and LV-EECs. production of PGI2 was significantly inhibited from cells exposed to 8-(dimetilamino) octyl 3,4,5-trymethoxybenzoate hydrochloride (10 and 100 microM: an inhibitor of intracellular calcium mobilization) or cyclopiazonic acid (10 microM: an endoplasmic reticulum Ca2+-ATPase inhibitor). These results indicate that shear stress enhances PGI2 release from cultured EECs and that mechanotransduction of shear stress depends on calcium mobilization in EECs.

Effect of aging on plasma membrane fluidity of rat aortic endothelial cells.

To assess age-related changes in the physical properties of vascular endothelial cell (EC) plasma membranes, we measured membrane fluidity with 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene, and 10-(1-pyrene)dodecanoic acid, and investigated the parameters affecting membrane fluidity of endothelial cells (ECs) cultured from the thoracic aortas of young (5-week-old) and aged (100-week-old) rats. Plasma membrane fluidity of aged rat ECs was significantly lower than that of young rat ECs, as assessed by increased 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization and by decreased pyrene excimer formation, although 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene did not demonstrate a change in membrane fluidity with aging. Compared with those in young rat ECs, cholesterol concentrations in aged rat ECs were significantly higher, whereas phospholipid concentrations were unchanged; consequently, the cholesterol/phospholipid molar ratio was significantly higher in aged rat ECs. Lipid peroxide levels measured with thiobarbituric acid reactive substances were significantly higher in EC plasma membranes of aged rats. These results indicate that age-related increases in cholesterol and lipid peroxide in vascular EC plasma membranes reduce membrane fluidity.

Maternal circulating nitrite levels are decreased in both normal normotensive pregnancies and pregnancies with preeclampsia.

OBJECTIVE: To evaluate whether maternal nitric oxide synthesis in pregnancies with preeclampsia is different from that in normal normotensive pregnancies. MATERIALS: Maternal circulating combined nitrate and nitrite levels or nitrite level were compared between 10 normotensive nonpregnant women, 30 normotensive pregnant women (10 first-trimester, 10 second-trimester, and 10 third-trimester pregnancies), 20 normotensive postpartum women (10 at 1 week after delivery, and 10 at 4 weeks after delivery), and 13 preeclamptic women (32 to 40 weeks' gestation). End-products of nitric oxide synthesis were measured from maternal venous blood samples using a fluorometric assay. RESULTS: Maternal circulating nitrite levels in nonpregnant women (1.13 +/- 0.22 microM) were significantly higher than those in the first-trimester pregnant women (0.68 +/- 0.13 microM), second-trimester pregnant women (0.65 +/- 0.13 microM), third-trimester pregnant women (0.48 +/- 0.17 microM), first puerperal week women (0.36 +/- 0.16 microM), and fourth puerperal week women (0.67 +/- 0.17 microM), respectively (p < 0.05). Maternal circulating nitrite level was decreased with advancing gestation, still remained low just after delivery, and was increased 4 weeks later. There was no significant difference in maternal circulating nitrite level between preeclamptic women (0.40 +/- 0.17 microM) and third-trimester pregnant women (0.48 +/- 0.17 microM). However, there were no significant differences in maternal circulating combined nitrate and nitrite levels among the groups. CONCLUSION: These results suggest that the maternal nitric oxide synthesis is not changed in normal normotensive pregnancies and pregnancies with preeclampsia. However, plasma nitrite level, which has stronger spasmolytic activity than the activity of the nitrate, was decreased in both normal normotensive pregnancies and pregnancies with preeclampsia.

Nitric oxide synthesis is increased after dehydroepiandrosterone sulphate administration in term human pregnancy.

The purpose of this study was to evaluate the role of nitric oxide in the vasodilative effect of dehydroepiandrosterone sulphate (DHEA-S) in term pregnant women. Circulating nitrite, nitrate and oestradiol concentrations were measured on 10 normal full-term pregnant women before (-30 min) and after (10, 30, 60, 90 and 120 min) administration of a 200 mg i.v. dose of DHEA-S dissolved in 20 ml of 5% dextrose (DHEA-S group). Ten normal full-term pregnant women received 20 ml of 5% dextrose as controls (control group). Maternal blood pressure and heart rate were also recorded. The median oestradiol concentration increased significantly after the infusion in DHEA-S group (P < 0.001), whereas there was no significant change in plasma oestradiol in the control group. In the DHEA-S group, plasma circulating nitrate and nitrite increased significantly at 10 and 30 min after DHEA-S administration respectively (P < 0.05). In the control group, there was no change in plasma nitric oxide (NO) metabolites. No change was found in heart rate or mean arterial blood pressure in the control or DHEA-S groups. These results suggest there may be a link between increased NO and increased oestrogen after DHEA-S injection but their peak values did not coincide. Both may be associated with vasodilation in term pregnant women.

Chronic administration of docosahexaenoic acid improves reference memory-related learning ability in young rats.

Wistar rats were fed a fish oil-deficient diet through three generations. The young (five-week-old) male rats of the third generation were randomly divided into two groups. Over 10 weeks, one group was perorally administered docosahexaenoic acid dissolved in 5% gum Arabic solution at 300 mg/kg/day; the other group received a similar volume of vehicle alone. Five weeks after starting the administration, the rats were tested for learning ability related to two types of memory, reference memory and working memory, with the partially (four of eight) baited eight-arm radial maze. Reference memory is information that should be retained until the next trial. Working memory is information that disappears in a short time. Entries into unbaited arms and repeated entries into visited arms were defined as reference memory errors and working memory errors, respectively. Docosahexaenoic acid administration over 10 weeks significantly reduced the number of reference memory errors, without affecting the number of working memory errors, and significantly increased the docosahexaenoic acid content and the docosahexaenoic acid/arachidonic acid ratio in both the hippocampus and the cerebral cortex. In addition, the ratio demonstrated a significantly negative correlation with the number of reference memory errors. These results suggest that chronic administration of docosahexaenoic acid is conducive to the improvement of reference memory-related learning ability, and that the docosahexaenoic acid/arachidonic acid ratio in the hippocampus or the cerebral cortex, or both, may be an indicator of learning ability.

Left atrial endocardium and prostacyclin.

In patients with rheumatic mitral stenosis, intracardiac thrombi are found mostly, for reasons still unknown, in the left atrium. We compared the release of PGI2 from the endocardium of the left atrium with that of the right ventricle and from the endothelium of the pulmonary arteries. Endocardial endothelial cells (EECs) were isolated from right ventricles (RV) and left atrial appendages (LAA) of porcine hearts, and vascular endothelial cells (VECs) from pulmonary arteries (PA) were obtained from the same animals. Cultured EEC and PA-VEC monolayers were placed in a pressure loading apparatus and incubated for 30 min under various pressures. After incubation, the supernatants were sampled and the 6-keto-PGF1 alpha contents measured. PGI2 release from LAA-EEC was much less than from RV-EEC or from PA-VEC. Moreover, transmural pressure did not enhance PGI2 release from LAA-EEC, although it did from RV-EEC and PA-EEC in a pressure-dependent manner. These results may explain why the left atrium is a common site for intracardiac thrombus formation in patients with mitral valve disease.

Age-related changes in platelet microviscosity and aggregation in rats.

1. To study possible changes in platelet microviscosity in aged animals, 1,6-diphenyl-1,3,5-hexatriene (DPH) was used as a nonpolar probe embedded in thrice-washed platelets from young, adult and aged rats. With the known values of maximum limiting anisotropy and the structural parameter of DPH and by estimating the steady state of fluorescence anisotropy and the average fluorescence of lifetime, we applied the Perrin equation to calculate the microviscosity. 2. We measured platelet aggregation, platelet lipids and platelet polyunsaturated fatty acids to determine any causal relationship between these parameters. Platelet aggregation, the platelet molar ratio of cholesterol to phospholipid ([C]/[PL]) and platelet microviscosity increased with age (P < 0.05) and were correlated with one another (P < 0.05). 3. Age-dependent increases in the steady state of fluorescence anisotropy, order parameters and the short component of fluorescence lifetime of the fluorophore were expressed as functions of variables, such as microviscosity or the [C]/[PL] ratio. 4. Platelet concentrations of arachidonic and eicosapentaenoic acids increased with age, but were not associated with aggregation. Age-related changes in microviscosity and the [C]/[PL] ratio seemed to be determinants affecting biophysical properties of platelet aggregation.

Impairment of serotonin-mediated nitric oxide release across the coronary bed in patients with coronary spastic angina.

Simultaneous assessment of plasma nitrate/nitrite and serotonin levels revealed possible impairment of serotonin-mediated nitric oxide release in patients with coronary spastic angina.

Association of age-related decrease in platelet membrane fluidity with platelet lipid peroxide.

The influence of age on platelet lipid peroxide (LPO), platelet membrane fluidity and the composition of fatty acid was investigated in female Wistar rats widely ranging in age from 14 to 720 days old. LPO levels were significantly higher (p<0.05) in the platelets of upper age groups than in those of lower age groups, showing a significantly positive correlation with age (r=0.84, p<0.0001). Membrane fluidity, assessed by 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence polarization, was significantly reduced with age. The composition of fatty acid demonstrated an age-related elevation (p<0.05) in the unsaturation index. The rises in the LPO levels revealed a significantly positive correlation with DPH-polarization (r=0.73, p<0.0001). Thus our results suggested that the age-related deterioration of platelet membrane fluidity, despite a significant elevation in the unsaturation index, was due to the age-related higher basal levels of LPO in platelets.

Hypotension induced by exercise is associated with enhanced release of adenyl purines from aged rat artery.

To determine whether the antihypertensive effects of exercise are associated with release of ATP and its metabolites from arteries, we assayed blood pressure and the release of adenine nucleotides and nucleosides from the caudal arteries of exercised and sedentary aged hypercholesterolemic rats. Exercise on a treadmill for 12 wk significantly decreased the rise in systolic and diastolic blood pressure by 7.5 and 15.9%, respectively, with advanced age. The concentrations of oleic, linoleic, and linolenic acids in the caudal artery decreased significantly with exercise, demonstrating an association between exercise and the unsaturation index of caudal arterial fatty acids. The amounts of total adenyl purines released by the arterial segments from exercised rats, both spontaneously and in response to norepinephrine, were significantly greater by 80.0 and 60.7%, respectively, than those released by tissues from sedentary rats. These results suggest that exercise alters the membrane fatty acid composition in aged rats as well as the release of ATP from vascular endothelial cells and that these factors are associated with the regression of the rise in blood pressure normally observed with advanced age.

Antioxidative effects of docosahexaenoic acid in the cerebrum versus cerebellum and brainstem of aged hypercholesterolemic rats.

Female Wistar rats (100 weeks old) were divided into two groups; one group was fed a high-cholesterol diet (HC) and the other a high-cholesterol diet plus docosahexaenoic acid (HC-fed DHA rats). Fatty acid concentrations in brain tissues were analyzed by gas chromatography. In the HC-fed DHA rats, brain catalase (CAT), GSH, and glutathione peroxidase (GPx) increased in the cerebrum but not in the brainstem or cerebellum. The rate of increase was 23.0% for CAT, 24.5% for GSH, and 26.3% for GPx compared with that in the HC animals (p < 0.05). In the cerebrum of the HC-fed DHA rats, CAT and GPx increased, with an increase in the ratio of DHA to arachidonic acid. The cerebrum, unlike the other areas of the brain, seems to be more sensitive to DHA in stimulating CAT and GPx. We suggest that DHA plays an important role in inducing an antioxidative defense against active oxygen by enhancing the cerebral activities of CAT, GPx, and GSH.

The hypotensive effect of docosahexaenoic acid is associated with the enhanced release of ATP from the caudal artery of aged rats.

Fish oils have been shown to lower blood pressure in hypertensive subjects. To determine the mechanism of this hypotensive effect, we examined the effects of docosahexaenoic acid (DHA), one of the (n-3) polyunsaturated fatty acids in fish oil, on blood pressure and on the release of adenyl purines, such as ATP, ADP, AMP and adenosine, from the caudal arteries of aged rats. Aged female Wistar rats (100 wk) were fed a high cholesterol diet and were administered intragastrically ethyl all-cis-4,7,10,13,16,19-docosahexaenoate [300 mg/(kg.d)] for 12 wk (DHA group) or vehicle alone (control group). Compared with the controls, rats supplemented with DHA had significantly greater (10.1%) DHA concentrations in the caudal arteries. This was associated with more total (n-3) arterial fatty acids, a greater unsaturation index of arterial fatty acids, 43.9% lower plasma noradrenaline levels and the repression of the elevation in blood pressure observed with advancing age. The amount of purines released, both spontaneously and in response to noradrenaline, from arterial segments of DHA-supplemented rats was significantly higher than that released from tissues of control rats. Regression analysis revealed significant negative relationships between the total amount of purines released from the artery and the systolic (SBP) and diastolic (DBP) blood pressures. These results suggest that in aged rats, supplementation with DHA alters the membrane fatty acid composition as well as the amount of ATP released from vascular endothelial cells and decreases plasma noradrenaline, and that these factors may ameliorate the rise in blood pressure normally associated with advancing age.

Effects of eicosapentaenoic acid and docosahexaenoic acid on plasma membrane fluidity of aortic endothelial cells.

We investigated the relative effects of n-3 eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) on the plasma membrane fluidity of endothelial cells (EC) cultured from the thoracic aorta by determining fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its cationic derivative trimethylamino-DPH (TMA-DPH). Fluidity assessed by TMA-DPH demonstrated no significant differences in plasma membranes of vehicle (dimethyl sulfoxide; DMSO)-, EPA-, and DHA-treated EC. Plasma membrane fluidity assessed by DPH polarization, however, was significantly higher in the order of DHA > EPA > DMSO. Total cholesterol content decreased significantly by 28.4 and 15.9% in the plasma membranes of DHA- and EPA-treated cells, respectively. Total phospholipid content remained unaltered in the plasma membranes of the three groups of cells; however, the molar ratio of total cholesterol to phospholipid decreased significantly only in the membranes of DHA-treated EC. The unsaturation index in the plasma membranes of EPA- and DHA-treated cells increased by 35.7 and 64.3%, respectively, compared with that in the plasma membranes of control cells. The activities of catalase and glutathione peroxidase in the whole-cell homogenates, and levels of lipid peroxides in either the whole-cell homogenates or in plasma membrane fractions were not altered in EPA- or DHA-treated EC. These results indicate that the influence of DHA is greater than that of EPA in increasing plasma membrane fluidity of vascular EC. We speculate that the greater effect of DHA compared to EPA is due to its greater ability to decrease membrane cholesterol content or the cholesterol/phospholipid molar ratio, or both, and also to its greater ability in elevating the unsaturation index in the plasma membranes of EC.

Long-term supplementation with a high cholesterol diet decreases the release of ATP from the caudal artery in aged rats.

We examined the effects of high cholesterol (HC) diet on the spontaneous and noradrenaline-induced release of ATP, ADP, AMP and adenosine from caudal arteries and on the plasma levels of these adenyl purines in aged (100-week-old) Wistar rats. Administration of this diet for 12 weeks significantly reduced spontaneous and noradrenaline (1 micromol/L)-evoked release of adenyl purines from the caudal arteries relative to rats given the control diet The unsaturation index of fatty acids (UI), which gives the average number of double bonds, of both the plasma and the caudal artery was significantly less in the HC diet-fed rats than in those fed the control diet. The HC diet for 12 weeks produced a slight but significant increase in systolic and diastolic blood pressure with advancing age. Regression analysis revealed a significant inverse relationship between the total amount of purines released from the artery and diastolic blood pressure, and also a positive relationship between the total amount of purines released and the UI of the caudal artery. These results suggest that the high cholesterol diet decreased the release of adenyl purines from the caudal arteries of aged rats, leading to an increase in blood pressure.

Age-related changes in aortic sensitivity to noradrenaline and acetylcholine in rats.

1. The purpose of the present study was to determine the relationship between plasma and tissue lipid levels and the effects of age on vascular responses to noradrenaline (NA) and acetylcholine (ACh). 2. Studies were performed in young and aged rats and the response of endothelium-intact and -denuded aortic rings to NA and to ACh was measured. The plasma concentration of cholesterol (total, high-density lipoprotein (HDL) and low-density lipoprotein (LDL)) and 17 beta-oestradiol was determined, as was the aortic tissue content of phospholipids, cGMP and cholesterol (total, free and esterified). 3. Levels of all types of cholesterol in plasma and aorta increased with age; cholesterol levels in plasma correlated with those in the aorta; levels of phospholipid in the aorta did not increase with age but correlated with those of LDL cholesterol in plasma; levels of 17 beta-oestradiol did not change, but those of cGMP increased with age. 4. In endothelium-intact rings, the maximum tension developed by exposure to NA did not change, but the EC50 of NA increased with age and correlated with total cholesterol in the plasma and with the levels of all types of cholesterol in the aorta. In rings precontracted with NA, age decreased the maximum relaxation induced by ACh. The EC50 of ACh decreased with age and was inversely correlated with levels of cholesterol in the plasma and aorta. Treatment with NA increased cGMP levels in aged rats. Removal of the endothelium abolished the response to ACh and heightened the sensitivity to NA in young and aged rats. 5. Aortic endothelial cells seem to inhibit amine-induced contraction, while age-related changes in the levels of cholesterol in aortic tissue affect the sensitivity of the tissue to NA and ACh.

Beneficial effects of FK409, a novel nitric oxide donor, on reperfusion injury of rat liver.

BACKGROUND: Nitric oxide (NO) seems to play an important role in modulating tissue injury during reperfusion of the liver. In this study, we have evaluated and compared the effects of FK409 (FK), a potent spontaneous NO releaser, and L-arginine in ischemia-reperfusion injury of the rat liver. METHODS: Male Sprague-Dawley rats underwent 90 min of hepatic ischemia followed by reperfusion. FK or L-arginine was used (intravenously) in two different doses for each drug (group I, 3.2 mg/kg FK; group II, 1.6 mg/kg FK; group IV, 100 mg/kg L-arginine; and group V, 300 mg/kg L-arginine). Saline was used in control animals (group III). Hepatic enzyme status, microcirculation, serum nitrite (NO2-) and nitrate (NO3-) and tissue injury score were evaluated at predetermined times. RESULTS: Serum NO2-/NO3- was elevated immediately by FK treatment dose-dependently but not by L-arginine. However, L-arginine caused late (6-24 hr) elevation of the NO metabolites dose-dependently. The elevation of serum aspartate aminotransferase and alanine aminotransferase was suppressed and hepatic microcirculation was improved in the FK-treated groups dose-dependently. L-Arginine also improved the microcirculation, but hepatic enzymes at 24 hr of reperfusion were significantly higher in group V than in the control group. These findings were well reflected by the extent of tissue injury in respective groups. CONCLUSION: FK treatment in the immediate reperfusion period improves hepatic microcirculation and confers a significant protective effect on hepatic ischemia-reperfusion injury in the rat.