BIOCHEMICAL SOCIETY TRANSACTIONS
1700
COM M U N ICATIONS Substrate Effects in Prostaglandin Biosynthesis in Vesicular Membranes CORNELIUS F. HARRINGTON, CATHERINE TRITSCHLER, ASUNCION McGRATH, THOMAS J. WALLEY and MICHAEL G. HARRINGTON Department of Biochemistry, University College, Beljield, Dublin 4, Ireland and JAMES F. CONNOLLY An Foras Taluntais, Animal Research Centre, Dunsinea, Co. Dublin, Ireland Prostaglandin precursor acids are derived from dietary linoleic acid by chain elongation and desaturation and are located in the membrane phospholipids, from which they are released by the action of phospholipase A, (Flower & Blackwell, 1976). Enzymic biosynthesis of individual prostaglandins in seminal-vesicular microsomal preparations may be directed specifically to production of prostaglandin E, by choice of substrate and cofactor constituents in the incubation medium (Takeguchi et a/., 1971). In the present study possible effects of dietary supplementation with linoleic acid on endogenous concentrations of prostaglandins and of prostaglandin E, synthesis by isolated microsomal fractions were examined. Animal seminal vesicles were removed as soon as possible after slaughter and were processed immediately or stored at -20°C before processing. Diced tissues were homogenized in the Polytron macerator in 4vol. of 100mM-Tris/HC1buffer, pH8.2, at 0 4 ° C . The microsomal fraction was isolated and incubated (37°C) with arachidonic acid, adrenaline (4.5 m ~ and ) reduced glutathione (4.5 mM). The incubation was terminated with ~ M - H C and ~ extracted with chloroform-methanol (2 :1, v/v). Prostaglandin E2 in theextract was determined by using AAzTsafter conversion into prostaglandin B, with KOH in methanol (Bygdeman & Samuelsson, 1964). The identity of the prostaglandins was confirmed by using t.1.c. (Nugteren et a[., 1966). Sheep vesicular microsomal fraction showed 70 %, bovine microsomal fraction 30 % and pig microsomal fraction less than lo:/, conversion of added arachidonic acid into prostaglandin E, under optimal conditions. The reaction showed an absolute requirement for both adrenaline and glutathione, was linear up to IOmin, occurred more rapidly, but with similar total percentage substrate conversion. at 37°C than at 30°C, and was subject to substrate inhibition at arachidonic acid concentrations greater than 0 . 1 6 ~ 1 ~ . This effect was previously reported by Yoshimoto et al. (1970) and Flower et al. (1973). Identical critical concentrations were observed with microsomal fractions from all three animal preparations tested, and were apparently independent of the enzyme concentration and its observed activity. Smith & Lands (1972) attributed this independence to inactivation of the synthetase complex by peroxide intermediates formed in the process. The possibility that the inhibitory effect was due to the detergent action of arachidonic acid on the integrity of the microsomal-membrane system appears to be substantiated by the following observations on the effects of high concentrations of arachidonic acid on the stability of the erythrocyte. Rabbit erythrocytes suspended in lOomM-Tris/ HCI buffer, pH8.2, underwent 20% lysis, which was prevented only when the Tris/HCI concentration was increased to 2 0 0 m ~ The . presence of 0 . 0 4 m ~ and - 00.8mM-arachidonic acid slightly lowered lysis in lOOmM-Tris/HCI buffer, whereas at both concentrations of Tris/HCl, 0.16m~~arachidonic acid caused 50% lysis and 0 . 3 2 m ~ -and 0.64m~-arachidonicacid caused 100% lysis of rabbit erythrocytes. The use of KCI to increase the osmoticity of the suspending medium did not alter the lytic effect in the erythrocyte, and had no effect on prostaglandin E2 synthesis in microsomal preparations. By taking advantage of the oesophageal-groove reflex in weanling calves, it was possible to increase significantly the polyunsaturated fatty acid: saturated fatty acid ratio in tissue lipid fractions (Connolly et a/., 1973). Seminal-vesicle preparations from 1977
571st MEETING, DUBLIN
1701
Table 1. prostaglandin E2 in seminal-vesicufar microsomal fractions from control and linoleic acid-supplementedveal calves Prostaglandin Ez concentrations were determined in extracts in 4.0ml of 100mM-Tris/ HCI, pH8.2, containing arachidonic acid (0.16m~),adrenaline (4.5 mM) and glutathione (4.5 mM). Results are means+s.E.M. (ten animals). Prostaglandin content (pg/g wet wt. of tissue) Preincubation 1.3k0.3 1.3k0.3
Control calves Linoleic acid-fed calves
Postincubat ion 5.3 f 0.2 5.4k0.3
Table 2. Prostaglandin E2synthesis in homogenatesprepared from seminal vesicles, lung and kidney medullafrom guinea pigsgroupedaccording to live weight (three animals in each group); 15min incubation at 37°C Wt. range (g) Mean wt. (9) Seminal vesicle Tissue wt. (g) Prostaglandin Ez produced (pg/total tissue) Lung Tissue wt. (g) Prostaglandin El produced (pg/total tissue) Kidney medulla Tissue wt. (g) Prostaglandin Ez produced (pgltotal tissue)
... ...
20040 270
400-600 570
600-800 690
800-1000 970
0.4 26.8
2.3 115
2.3 139
5.3 28 1
3.0 105
4.3 25 1
4.9 525
7.2 410
2.0 84.2
2.5 178
2.7 252
3.8 212
animals receiving a linoleic acid-supplemented diet for 6 weeks before slaughter were compared with similar preparations from unsupplemented pair-fed control animals. Polyunsaturated fatty acids, mainly linoleic acid, were shown by g.1.c. to be increased 2-fold in the microsomal fraction prepared from seminal vesicles of linoleic acidsupplemented animals. Endogenous prostaglandin concentrations and prostaglandin E2 synthetic ability of such microsomal fractions (Table 1) showed no differences from those of control animals. In preparations from both groups of animals, synthesis of prostaglandin Ez was considerably less than that observed in experiments using tissues obtained from the commercial abattoir. Because of the constraints imposed when using this dietary regime, in particular in relation to the age and hence the immaturity of the animals involved, seminal vesicles may not be a useful indicator of possible effects of alteration by dietary means of tissue precursors of prostaglandin synthesis. The extent of prostaglandin Ez synthesis in homogenates and microsomal fractions from seminal vesicles, lung and kidney medulla from guinea pigs at selected stages of development was therefore determined. Animals were arbitrarily grouped according to live weights in the ranges 200-400,400-600,600-800 and 800-1OOOg. These groups were selected to correspond to the age of puberty, early reproductive, late reproductive and aged phases of the life of the animal. The results (Table 2) show considerable variation in prostaglandin Ez synthetase activity in seminal vesicles with age, being extremely low at puberty. In lung tissue, activity appears to increase with age of animal until the late adult phase, decreasing in the aged animal. In kidney medulla, a similar increase in activity up to the late adult phase was recorded, with little or no change in the aged animals.
VOl. 5
BlOCH EM ICAL SOCIETY TRANSACTIONS
I702
The support of the Medical Research Council of Ireland is acknowledged. T. J. W. was the holder of an M.R.C. Vacation Studentship. C. F. H. is grateful to An Foras Taluntais for a postgraduate award. We are grateful to Dr. J. E. Pike (Upjohn) for generously giving the prostaglandins. Bygdeman, M. & Samuelsson B. (1964) Clin. Chim. Acra, 10,569-571 Connolly, J. F., Noonan, M. & Harrington, M. G. (1973) Ahstr. Spec. Meet. FEBSno. 171 Flower, R. J. & Blackwell, G. J. (1976) Biochem. Pharmacol.25,285-291 Flower, R. J., Cheung, H. S. & Cushman, D. W. (1973)Prosfaglandins 4,325-341 Nugteren, D. H., Beerthuis, R. K. &van Dorp, D. A. (1966) Receuil85,405-419 Smith, W. L. &Lands W. E. M. (1972) Biochemistry 11, 3276-3285 Takeguchi, C., Kohno, E. & Sih, C. J. (1971) Biochemistry 10,2372-2376 Yoshimoto, A., Ito, H. & Tomita, K. (1970)J. Biochem. (Tokyo) 68,487499
Linoleic Acid Metabolism in Normal and Acutely I11 Rats CORNELIUS F. HARRINGTON, PATRICK T. S. MA and W. CLAYTON LOVE Clinical Biochemistry Laboratory, Department of Biochemistry, Trinity College, Dublin 2 , Ireland
Serum linoleic acid concentrations are decreased in multiple sclerosis (Baker et ul., 1964; Tichy et al., 1969) and in a variety of acute illnesses (Love et al., 1973). In multiple sclerosis and acute non-neurological illnesses, the decreased linoleate content is associated with increased oleic acid, palmitic acid and palmitoleic acid content (Love et al., 1972, 1973, 1974). Dietary intake (Callaghan et al., 1973) and absorption of linoleate (Belin et al., 1968) is not decreased in multiple-sclerosis patients compared with healthy controls. The decrease in plasma linoleate appears to be proportional to the severity of the illness in both multiple sclerosis (Love et al., 1974) and non-neurological disease (Callaghan et al., 1976). As intake and absorption of linoleate are normal, at least in multiple sclerosis, metabolism of this acid must be altered in acute illness, since plasma content is decreased. The aim of the present study was to establish a reproducible illness in rats in which linoleate metabolism could be compared with healthy controls.
Table I. Percentage distribution of lipid radioactivity atnong subcellular fractions of rut tissues 3 h after injection of [‘4C]linoleicacid into the femoral vein Distribution (%)
Tissue Normal rat Brain Heart Kidney Liver Muscle Anaemic rat Brain Heart Kidney Liver Muscle
Total radioactivity (c.p.m.)
PostMitochondria
Microsomal fraction
microsomal supernatant
1740 6090 5060 18840 1770
66 71 55 49 31
10 11 16 19 27
24 18 29 32 42
2210 6540 7560 26 300 1980
67 49 42 49 43
5 31 27 5 28
28 20 31 46 29
Fraction
...
1977
1700
COM M U N ICATIONS Substrate Effects in Prostaglandin Biosynthesis in Vesicular Membranes CORNELIUS F. HARRINGTON, CATHERINE TRITSCHLER, ASUNCION McGRATH, THOMAS J. WALLEY and MICHAEL G. HARRINGTON Department of Biochemistry, University College, Beljield, Dublin 4, Ireland and JAMES F. CONNOLLY An Foras Taluntais, Animal Research Centre, Dunsinea, Co. Dublin, Ireland Prostaglandin precursor acids are derived from dietary linoleic acid by chain elongation and desaturation and are located in the membrane phospholipids, from which they are released by the action of phospholipase A, (Flower & Blackwell, 1976). Enzymic biosynthesis of individual prostaglandins in seminal-vesicular microsomal preparations may be directed specifically to production of prostaglandin E, by choice of substrate and cofactor constituents in the incubation medium (Takeguchi et a/., 1971). In the present study possible effects of dietary supplementation with linoleic acid on endogenous concentrations of prostaglandins and of prostaglandin E, synthesis by isolated microsomal fractions were examined. Animal seminal vesicles were removed as soon as possible after slaughter and were processed immediately or stored at -20°C before processing. Diced tissues were homogenized in the Polytron macerator in 4vol. of 100mM-Tris/HC1buffer, pH8.2, at 0 4 ° C . The microsomal fraction was isolated and incubated (37°C) with arachidonic acid, adrenaline (4.5 m ~ and ) reduced glutathione (4.5 mM). The incubation was terminated with ~ M - H C and ~ extracted with chloroform-methanol (2 :1, v/v). Prostaglandin E2 in theextract was determined by using AAzTsafter conversion into prostaglandin B, with KOH in methanol (Bygdeman & Samuelsson, 1964). The identity of the prostaglandins was confirmed by using t.1.c. (Nugteren et a[., 1966). Sheep vesicular microsomal fraction showed 70 %, bovine microsomal fraction 30 % and pig microsomal fraction less than lo:/, conversion of added arachidonic acid into prostaglandin E, under optimal conditions. The reaction showed an absolute requirement for both adrenaline and glutathione, was linear up to IOmin, occurred more rapidly, but with similar total percentage substrate conversion. at 37°C than at 30°C, and was subject to substrate inhibition at arachidonic acid concentrations greater than 0 . 1 6 ~ 1 ~ . This effect was previously reported by Yoshimoto et al. (1970) and Flower et al. (1973). Identical critical concentrations were observed with microsomal fractions from all three animal preparations tested, and were apparently independent of the enzyme concentration and its observed activity. Smith & Lands (1972) attributed this independence to inactivation of the synthetase complex by peroxide intermediates formed in the process. The possibility that the inhibitory effect was due to the detergent action of arachidonic acid on the integrity of the microsomal-membrane system appears to be substantiated by the following observations on the effects of high concentrations of arachidonic acid on the stability of the erythrocyte. Rabbit erythrocytes suspended in lOomM-Tris/ HCI buffer, pH8.2, underwent 20% lysis, which was prevented only when the Tris/HCI concentration was increased to 2 0 0 m ~ The . presence of 0 . 0 4 m ~ and - 00.8mM-arachidonic acid slightly lowered lysis in lOOmM-Tris/HCI buffer, whereas at both concentrations of Tris/HCl, 0.16m~~arachidonic acid caused 50% lysis and 0 . 3 2 m ~ -and 0.64m~-arachidonicacid caused 100% lysis of rabbit erythrocytes. The use of KCI to increase the osmoticity of the suspending medium did not alter the lytic effect in the erythrocyte, and had no effect on prostaglandin E2 synthesis in microsomal preparations. By taking advantage of the oesophageal-groove reflex in weanling calves, it was possible to increase significantly the polyunsaturated fatty acid: saturated fatty acid ratio in tissue lipid fractions (Connolly et a/., 1973). Seminal-vesicle preparations from 1977
571st MEETING, DUBLIN
1701
Table 1. prostaglandin E2 in seminal-vesicufar microsomal fractions from control and linoleic acid-supplementedveal calves Prostaglandin Ez concentrations were determined in extracts in 4.0ml of 100mM-Tris/ HCI, pH8.2, containing arachidonic acid (0.16m~),adrenaline (4.5 mM) and glutathione (4.5 mM). Results are means+s.E.M. (ten animals). Prostaglandin content (pg/g wet wt. of tissue) Preincubation 1.3k0.3 1.3k0.3
Control calves Linoleic acid-fed calves
Postincubat ion 5.3 f 0.2 5.4k0.3
Table 2. Prostaglandin E2synthesis in homogenatesprepared from seminal vesicles, lung and kidney medullafrom guinea pigsgroupedaccording to live weight (three animals in each group); 15min incubation at 37°C Wt. range (g) Mean wt. (9) Seminal vesicle Tissue wt. (g) Prostaglandin Ez produced (pg/total tissue) Lung Tissue wt. (g) Prostaglandin El produced (pg/total tissue) Kidney medulla Tissue wt. (g) Prostaglandin Ez produced (pgltotal tissue)
... ...
20040 270
400-600 570
600-800 690
800-1000 970
0.4 26.8
2.3 115
2.3 139
5.3 28 1
3.0 105
4.3 25 1
4.9 525
7.2 410
2.0 84.2
2.5 178
2.7 252
3.8 212
animals receiving a linoleic acid-supplemented diet for 6 weeks before slaughter were compared with similar preparations from unsupplemented pair-fed control animals. Polyunsaturated fatty acids, mainly linoleic acid, were shown by g.1.c. to be increased 2-fold in the microsomal fraction prepared from seminal vesicles of linoleic acidsupplemented animals. Endogenous prostaglandin concentrations and prostaglandin E2 synthetic ability of such microsomal fractions (Table 1) showed no differences from those of control animals. In preparations from both groups of animals, synthesis of prostaglandin Ez was considerably less than that observed in experiments using tissues obtained from the commercial abattoir. Because of the constraints imposed when using this dietary regime, in particular in relation to the age and hence the immaturity of the animals involved, seminal vesicles may not be a useful indicator of possible effects of alteration by dietary means of tissue precursors of prostaglandin synthesis. The extent of prostaglandin Ez synthesis in homogenates and microsomal fractions from seminal vesicles, lung and kidney medulla from guinea pigs at selected stages of development was therefore determined. Animals were arbitrarily grouped according to live weights in the ranges 200-400,400-600,600-800 and 800-1OOOg. These groups were selected to correspond to the age of puberty, early reproductive, late reproductive and aged phases of the life of the animal. The results (Table 2) show considerable variation in prostaglandin Ez synthetase activity in seminal vesicles with age, being extremely low at puberty. In lung tissue, activity appears to increase with age of animal until the late adult phase, decreasing in the aged animal. In kidney medulla, a similar increase in activity up to the late adult phase was recorded, with little or no change in the aged animals.
VOl. 5
BlOCH EM ICAL SOCIETY TRANSACTIONS
I702
The support of the Medical Research Council of Ireland is acknowledged. T. J. W. was the holder of an M.R.C. Vacation Studentship. C. F. H. is grateful to An Foras Taluntais for a postgraduate award. We are grateful to Dr. J. E. Pike (Upjohn) for generously giving the prostaglandins. Bygdeman, M. & Samuelsson B. (1964) Clin. Chim. Acra, 10,569-571 Connolly, J. F., Noonan, M. & Harrington, M. G. (1973) Ahstr. Spec. Meet. FEBSno. 171 Flower, R. J. & Blackwell, G. J. (1976) Biochem. Pharmacol.25,285-291 Flower, R. J., Cheung, H. S. & Cushman, D. W. (1973)Prosfaglandins 4,325-341 Nugteren, D. H., Beerthuis, R. K. &van Dorp, D. A. (1966) Receuil85,405-419 Smith, W. L. &Lands W. E. M. (1972) Biochemistry 11, 3276-3285 Takeguchi, C., Kohno, E. & Sih, C. J. (1971) Biochemistry 10,2372-2376 Yoshimoto, A., Ito, H. & Tomita, K. (1970)J. Biochem. (Tokyo) 68,487499
Linoleic Acid Metabolism in Normal and Acutely I11 Rats CORNELIUS F. HARRINGTON, PATRICK T. S. MA and W. CLAYTON LOVE Clinical Biochemistry Laboratory, Department of Biochemistry, Trinity College, Dublin 2 , Ireland
Serum linoleic acid concentrations are decreased in multiple sclerosis (Baker et ul., 1964; Tichy et al., 1969) and in a variety of acute illnesses (Love et al., 1973). In multiple sclerosis and acute non-neurological illnesses, the decreased linoleate content is associated with increased oleic acid, palmitic acid and palmitoleic acid content (Love et al., 1972, 1973, 1974). Dietary intake (Callaghan et al., 1973) and absorption of linoleate (Belin et al., 1968) is not decreased in multiple-sclerosis patients compared with healthy controls. The decrease in plasma linoleate appears to be proportional to the severity of the illness in both multiple sclerosis (Love et al., 1974) and non-neurological disease (Callaghan et al., 1976). As intake and absorption of linoleate are normal, at least in multiple sclerosis, metabolism of this acid must be altered in acute illness, since plasma content is decreased. The aim of the present study was to establish a reproducible illness in rats in which linoleate metabolism could be compared with healthy controls.
Table I. Percentage distribution of lipid radioactivity atnong subcellular fractions of rut tissues 3 h after injection of [‘4C]linoleicacid into the femoral vein Distribution (%)
Tissue Normal rat Brain Heart Kidney Liver Muscle Anaemic rat Brain Heart Kidney Liver Muscle
Total radioactivity (c.p.m.)
PostMitochondria
Microsomal fraction
microsomal supernatant
1740 6090 5060 18840 1770
66 71 55 49 31
10 11 16 19 27
24 18 29 32 42
2210 6540 7560 26 300 1980
67 49 42 49 43
5 31 27 5 28
28 20 31 46 29
Fraction
...
1977
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