IfROSTAGLANDINSLEUKOTRIENES ANDESSENTIALFATTYACIDS Prostaglandms Leukotrienes and Essential 0 Longman Group UK Ltd 1992
Fatty Acids (1992) 46.215-217
Production of Platelet Thromboxane A2 and Arterial Prostacyclin I2 from Hypercholesterolemic Rats J. H. Arnold, K. A. Pritchard Jr, N. J. Greco and R. V. Panganamala Department of Medical Biochemistry, (Reprint requests to RVP)
The Ohio State University, 1645 Neil Avenue, Columbus,
Ohio 43210, USA
ABSTRACT. The plasma cholesterol, plasma malonaldehyde (MDA), platelet thromboxane A2 (TXAJ and vascular prostacyclin (PGI,) were measured in male Sprague-Dawley rats fed diets supplemented with cholesterol (1%) and cholic acid (0.5%). For comparisons, measurements were made in rats fed normal diets. The concentration of cholesterol in the plasma of rats had reached a maximum in 1 week of feeding experimental diets. TXAz production from collagen and thrombin stimulated platelets was significantly decreased in animals fed experimental diets for 1 week. The production of MDA in the plasma of animals fed experimental diets for 8 weeks was significantly lower compared to the animals fed normal diets. There was a small but significant reduction in the formation of PGIz in rats fed experimental diets for 8 weeks. These data suggest that feeding cholesterol rich diets to rats alters the platelet membrane properties differently from human and rabbit. Furthermore, cholesterol feeding to rats had some damaging effect on the arterial PGIz synthesis.
Corp (Boston, MA). Standard TXB?_ and 6keto-PGF,, were kind gifts from Dr John Pike (Upjohn Co). Antisera for TXB, and 6-keto-PGF,, were kind gifts from Dr L. Levine (Brandeis University, Waltham, MA). Arachidonic acid was purchased from Nuchek Prep (Elysian, MN). Bovine tendon collagen and bovine thrombin were purchased from Sigma Chemical Co (St. Louis, MO). Animal diets were formulated by ICN (Cleveland, OH). The composition of the diets is shown in Table 1.
INTRODUCTION The effects of dietary cholesterol on the etiology of atherosclerosis have been studied in several animal models. In cholesterol rabbits, increased platelet sensitivity to aggregation agonists, increased thromboxane A, (TXA*) production and decreased arterial prostacyclin (PGI,) synthesis have been reported (1,2). Similar platelet sensitivity to aggregating agonists and increased TXA? production have been reported in human blood platelets when incubated with cholesterol-rich liposomes (3). Whereas rabbits are highly susceptible to cholesterol-induced atherosclerosis, the rat model has been proven relatively resistant to the development of atheromatous arterial lesion unless the diets are supplemented with agents such as thiouracil or bile salts (4). In this study, we report the effects of hypercholesterolemic diets on the rat platelet TXA, and arterial PGI, production.
MATERIALS
Dietary regimen Male Sprague-Dawley rats weighing 200-300 g were divided into two groups. One group was maintained on Table 1 Composition Components
Dextrin Casein Sucrose Coconut oil Salt mix USPXVII Liver concentrate Choline chloride DL-methionine Inositol Vitamin fortification Cholesterol Cholic acid
AND METHODS
Tritiated TXB2 (150 Ci/mmole) and 6-keto PGF,, (100 Ci/mmole) were purchased from New England Nuclear
Date received 26 November Date accepted 23 December
199 1 1991 215
of the experimental
and control diet
Control (g/IO kg diet)
1000 1560 1000 200 40 20 20 10 400 _
Experimental
4800 1000 1560 1000 200 40 20 20 10 400 100 50
216
Prostaglandins
Leukotrienes
and Essential Fatty Acids
the control diet and the other was maintained on the experimental diet. One group of control and experimental rats were sacrificed after 1 week on the diets and the second group of control and experimental rats were sacrificed after 8 weeks on the diets. Plasma cholesterol and triglycerides were determined by A-gent determinant using ABA100 Analyzer.
Table 3 Platelet TXB, synthesis in control and experimental Collagen (200 mg/ml)
Group
(pmoles/lOs
I Week (IO)
Control Experimental
8 Weeks (IO)
Control Experimental
Isolation of platelets and incubation procedures Rats were sacrificed by injection (ip.) of Napentabarbital (lg/ml). Blood was drawn from the heart and mixed with trisodium citrate (3.8%) in the ratio of 9: 1. Platelet rich plasma (PRP) was obtained by centrifugation on GLC-2 at 1500 rpm for 15 min. PRP was further centrifuged at 4000 rpm for 20 min on a RC-2 centrifuge. The resulting platelet pellet was resuspended in calcium-free Krebs buffer. Platelets were adjusted to 2 x lo* platelet per ml of suspending buffer. Platelets (1 x 10s) were incubated for 2 min with collagen (200 mg/ml) or thrombin (0.5 U/ml).
Determination of TXB, Platelet TXB2 was determined previously (6).
by RIA as described
Determination of 6-keto-PGF,, Aorta tissue was isolated as described elsewhere (6). Slices (10-12 mg) of aortic tissue were incubated with and without arachidonic acid. Aliquots of incubation were determined for 6-keto-PGF,, by RIA (6).
Estimation of lipid peroxides Lipid peroxides in the plasma were estimated as thiobarbuturic acid reacting substance (TBARS) by the method of Satoh et al (7).
Statistical analysis Significance of differences between means was determined using Student’s one-tailed t-test. Data is presented as mean f SEM, and the numbers in the parenthesis represent the number of animals in each group.
5.4 * 3.1 * p< 12.1 * s.3 * p
Fatty Acids (1992) 46.215-217
Production of Platelet Thromboxane A2 and Arterial Prostacyclin I2 from Hypercholesterolemic Rats J. H. Arnold, K. A. Pritchard Jr, N. J. Greco and R. V. Panganamala Department of Medical Biochemistry, (Reprint requests to RVP)
The Ohio State University, 1645 Neil Avenue, Columbus,
Ohio 43210, USA
ABSTRACT. The plasma cholesterol, plasma malonaldehyde (MDA), platelet thromboxane A2 (TXAJ and vascular prostacyclin (PGI,) were measured in male Sprague-Dawley rats fed diets supplemented with cholesterol (1%) and cholic acid (0.5%). For comparisons, measurements were made in rats fed normal diets. The concentration of cholesterol in the plasma of rats had reached a maximum in 1 week of feeding experimental diets. TXAz production from collagen and thrombin stimulated platelets was significantly decreased in animals fed experimental diets for 1 week. The production of MDA in the plasma of animals fed experimental diets for 8 weeks was significantly lower compared to the animals fed normal diets. There was a small but significant reduction in the formation of PGIz in rats fed experimental diets for 8 weeks. These data suggest that feeding cholesterol rich diets to rats alters the platelet membrane properties differently from human and rabbit. Furthermore, cholesterol feeding to rats had some damaging effect on the arterial PGIz synthesis.
Corp (Boston, MA). Standard TXB?_ and 6keto-PGF,, were kind gifts from Dr John Pike (Upjohn Co). Antisera for TXB, and 6-keto-PGF,, were kind gifts from Dr L. Levine (Brandeis University, Waltham, MA). Arachidonic acid was purchased from Nuchek Prep (Elysian, MN). Bovine tendon collagen and bovine thrombin were purchased from Sigma Chemical Co (St. Louis, MO). Animal diets were formulated by ICN (Cleveland, OH). The composition of the diets is shown in Table 1.
INTRODUCTION The effects of dietary cholesterol on the etiology of atherosclerosis have been studied in several animal models. In cholesterol rabbits, increased platelet sensitivity to aggregation agonists, increased thromboxane A, (TXA*) production and decreased arterial prostacyclin (PGI,) synthesis have been reported (1,2). Similar platelet sensitivity to aggregating agonists and increased TXA? production have been reported in human blood platelets when incubated with cholesterol-rich liposomes (3). Whereas rabbits are highly susceptible to cholesterol-induced atherosclerosis, the rat model has been proven relatively resistant to the development of atheromatous arterial lesion unless the diets are supplemented with agents such as thiouracil or bile salts (4). In this study, we report the effects of hypercholesterolemic diets on the rat platelet TXA, and arterial PGI, production.
MATERIALS
Dietary regimen Male Sprague-Dawley rats weighing 200-300 g were divided into two groups. One group was maintained on Table 1 Composition Components
Dextrin Casein Sucrose Coconut oil Salt mix USPXVII Liver concentrate Choline chloride DL-methionine Inositol Vitamin fortification Cholesterol Cholic acid
AND METHODS
Tritiated TXB2 (150 Ci/mmole) and 6-keto PGF,, (100 Ci/mmole) were purchased from New England Nuclear
Date received 26 November Date accepted 23 December
199 1 1991 215
of the experimental
and control diet
Control (g/IO kg diet)
1000 1560 1000 200 40 20 20 10 400 _
Experimental
4800 1000 1560 1000 200 40 20 20 10 400 100 50
216
Prostaglandins
Leukotrienes
and Essential Fatty Acids
the control diet and the other was maintained on the experimental diet. One group of control and experimental rats were sacrificed after 1 week on the diets and the second group of control and experimental rats were sacrificed after 8 weeks on the diets. Plasma cholesterol and triglycerides were determined by A-gent determinant using ABA100 Analyzer.
Table 3 Platelet TXB, synthesis in control and experimental Collagen (200 mg/ml)
Group
(pmoles/lOs
I Week (IO)
Control Experimental
8 Weeks (IO)
Control Experimental
Isolation of platelets and incubation procedures Rats were sacrificed by injection (ip.) of Napentabarbital (lg/ml). Blood was drawn from the heart and mixed with trisodium citrate (3.8%) in the ratio of 9: 1. Platelet rich plasma (PRP) was obtained by centrifugation on GLC-2 at 1500 rpm for 15 min. PRP was further centrifuged at 4000 rpm for 20 min on a RC-2 centrifuge. The resulting platelet pellet was resuspended in calcium-free Krebs buffer. Platelets were adjusted to 2 x lo* platelet per ml of suspending buffer. Platelets (1 x 10s) were incubated for 2 min with collagen (200 mg/ml) or thrombin (0.5 U/ml).
Determination of TXB, Platelet TXB2 was determined previously (6).
by RIA as described
Determination of 6-keto-PGF,, Aorta tissue was isolated as described elsewhere (6). Slices (10-12 mg) of aortic tissue were incubated with and without arachidonic acid. Aliquots of incubation were determined for 6-keto-PGF,, by RIA (6).
Estimation of lipid peroxides Lipid peroxides in the plasma were estimated as thiobarbuturic acid reacting substance (TBARS) by the method of Satoh et al (7).
Statistical analysis Significance of differences between means was determined using Student’s one-tailed t-test. Data is presented as mean f SEM, and the numbers in the parenthesis represent the number of animals in each group.
5.4 * 3.1 * p< 12.1 * s.3 * p
