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Increase in the formation of cyclooxygenase and lipoxygenase products in kidneys of adrenalectomized rats
Prostaglandins Leukotrienes and Medicine 24: 123-127, 1986
INCREASE IN THE FORMATION OF CYCLOOXYGENASE KIDNEYS OF ADRENALECTOMIZED RATS
AND
LIPOXYGENASE
PRODUCTS
IN
J.E. Vincent, F.J. Zijlstra and T.E. Gezel Department of Pharmacology, Faculty of Medicine Erasmus University Rotterdam, P.O.Box 1738 3000 DR Rotterdam, The Netherlands
ABSTRACT The effect of adrenalectomy on the formation of cyclooxygenase and lipoxygenase products from [1-14C]-arachidonic acid in rat kidneys after incubation with the Calcium-ibnophore A23187 has been determined. The metabolites were isolated by HPLC. The main components formed are PGD2, di-HETE and 5- ,12and 15-HETE. After PGE2. PGF2,, LTB4, 5 ,12 adrenalectomy, an increase occurs in the formation of PG and LT. which is highest in that of PGD2 and lP-HETE. These effects are most probably to a diminished formation or inactivation of lipocortin in related adrenalectomized animals, a glucocorticosteroid induced peptide with phospholipase A2 inhibitory activity. INTRODUCTION In rat kidneys, formation of metabolites of arachidonic acid 1) by both the cyclooxygenase and lipoxygenase pathways occurs. It has been reported that in this tissue PGD2, PGE2, PGF20, TxB2, 6-keto-PGF,o and 1 P-HETE are formed (1,2). In rat glomeruli, the main lipoxygenase product formed was 12-HETE (3). The initial reaction in the formation of these substances is the release of AA by the activation of P1A2. Recently, it has been shown that This in several types of cells an inhibitor of P1A2, lipocortin is present. substance has been isolated from macrophages, neutrophils and renomedullary interstitial formation is cells Its induced by (4,5,6). glucocorticosteroids. It has been demonstrated that in thymocytes, after lipocortin is inactivated by activation induced by several substances In this way, the inhibitor is no longer active and phosphorylation (7). P1A2 is activated, followed by the release of AA and the formation of metabolites. It is not known, whether the same mechanism exists in other 1)The following abbreviations were used: AA = arachidonic acid, LTs = leukotrienes, TxB2 = thromboxane B2, PGs = prostaglandins, 5- ,12- ,15- HETE = 5- ,12- ,15- hydroxy-6,8,11,14-eicosatetraenoic acid , P1A2 = diHETE = 5-.12-dihydroxy-6,8,11,14-eicosatetraenoic acid, phospholipase A2, adrex = adrenalectomized.
123
on the presence of is dependent cells. As the formation of lipocortin glucocorticosteroids, a decrease or inactivation may occur in adrex animals. In our experiments, the effect of adrenalectomy on the production of AA in rat kidneys after activation by the Calciumionophore A23187 metabolites was determined. MATERIALS AND METHODS were - Wistar strain male rats of 200g weight used. Animals was performed via a small medial incision. As drinking water Adrenalectomy a 1Z salt solution was given. Sham operated rats were used as controls, put The animals were used in the experiments several on normal drinking water. weeks after the operation. AA metobolite formation - The animals were sacrificed and the kidneys removed. The kidney weight was approx. lg. After chopping the tissue was incubated at 37'C and continuously gassed through a thin pipette with a mixture of 95% 02 and 5% COP. 2.5 uCi [1-14C] AA (55 mCi/mol) was added, luthathione (final cont. 2 mmol/l) and Ca-ionophore-A23187 (10 vmol/l), 4 H-LT and 'H-PG were added and the homogenate spun down (10 min, 14OOxg, respectively of 4OC). Serine and cysteine were added to prevent breakdown The pellets were washed once, and the LTC4 to LTD4 and LTD4 to LTE4. combined supernatants of each cell type were then applied to a couple of Sep-Pak C,8 and silica cartridges. The latter were prewashed with 10 ml ethanol and 10 ml distilled water. The samples were eluted with 5 ml ethanol on each column; these eluates were combined and evaporated to Thereafter, the dried dryness with a gentle stream of nitrogen at 37'C. in 0.5 ml solvent A (tetrahydrofuran-methanol-0.146 samples were dissolved EDTA in water-acetic acid, 25:30:45:0.1 to pH v/v, adjusted 5.5), centrifuged and purified by a Millex filter and kept in a HPLC microvial. Solvent system A was used for separation of lipoxygenase products on a Nucleosil 5C,8 column. The flow rate was 0.9 ml/min, the oven temperature 37Oc. Prostaglandins were separated on a Zorbax C8 column using system B (acetonitrile-benzene-water-acetic acid, 24:0.2:76:0.1 v/v), with a flow into the column and Volumes of 100 ,ul were injected rate of 2 ml/min. One minute chromatographed using a 1082B chromatograph (Hewlett Packard). The chromatographic fractions were collected for scintillation counting. method and the identification of the peaks have been described earlier (8).
PJateriols- Leukotriene B4 was a gift of Dr. J. Rokach (Merck Frosst A23187 was obtained from Hoechst, [1-14C] AA C ada Inc.). Ca-ionophore [Yn H(n)]5-HETE, ['H(n)] 12-HETE and C'H(nJ] 15-HETE were obtained from New ['H(n)] PGE2, [ H(n)]PGF20 and ['H(n)] PGD2 from England Nuclear and Sep-Pak C,8 and silica cartridges and HPLC filters, HA (0.45 urn), Amersham. FH (0.5 urn) and Millex (0.45 urn)were obtained from Waters/Millipore, HPLC microvials from Weidmann Plastics, Switzerland, Prepacked Nucleosil 5C18 and Zorbax C8 HPLC columns were obtained from Chrompack. RESULTS The substances formed from [1-14C] AA by the cyclooxygenase and lipoxygenase pathways in the kidneys after activation with the Ca-ionophore A23187 have been determined. In the control animals PGD2, POE2 and PGF2o were found. The substances LTB4, 5-HETE, 12-HETE and 15-HETE were present on the chromatogram as shown in fig. 1, representing a separation of lipoxygenase products. 124
(5S,12S)-5,12-dihydroxy-6,8,10 trons-14 identified as One peak was cis-eicosatetraenoic acid (M. Claeys, personal communication). The amounts The differences formed in .the sham operated rats are shown in table I. between the amounts formed by the control and adrex rats are represented in fig. 2. There were large increases in the formation of PGD2 and 12-HETE and smaller ones in the other products. 4000
3000
w f
2000
5 P
1000
10
20
30
40
Time
50
60
70
80
(min)
Fig. 1. Chromatogram of lipoxygenase products formed from [1-'4C] AA by chopped kidney tissue of a sham operated rat after stimulation with A23187.
Table
I.
Formation of eicosanoids from operated rats. (n=5)
[1-'4C] AA
dpm x 1000/g
gg2a
10.5 6.1
LTB2 PGD2
<0.2 5.7 f ?? 0.1 1.4
5 -i&c* 12-HETE 15-HETE
28.1 f 4.7 31.4 f 8.2 16.8 f 2.5
i
2.4 1.5
125
in
kidneys of
sham
I PGF2,
-
PGE2
T*
T*
1
L
-
PCD, HETE
HETE
A 5-
HETE
Fig. 2. Difference in formation of cyclooxygenase and lipoxygenase products formed from [l-14C] AA in kidneys of sham operated and adrenalectomized rats after activation by A23187. Data f SEM. *P
DISCUSSION of the In the kidney. the medulla is the main site of production These substances have been implicated in several aspects of prostaglandins. kidney function such as the regulation of renal blood flow, water and sodium filtration. PGD2 induces glomerular renin transport, release and It may prevent the aggregation. vasodilatation and inhibits platelet formation of platelet aggregates which can impair glomerular filtration. The role of the lipoxygenase derivatives in the kidney is not well known. The effects of LTB4 are chemotaxis and the induction of increased vascular The 5- and 12-HETEs also have small chemotactic effects. permeability. mineral0 After adrenalectomy, the secretion of glucocorticosteroids, corticosteroids and adrenaline will rapidly diminish and disappear. The absence of circulating steroids will lead to an increase in the secretion of How does this affect the metabolism of unsaturated fatty acids ? No ACTH. action of mineralocorticosteroids has yet been demonstrated on this factor. and ACTH both induce the lipolysis of adipose tissue, leading to Adrenaline the release of fatty acids. This effect is very small in the absence of glucocorticosteroids.
126
As has been mentioned before, the effects of adrenalectomy can mOSt probably be ascribed to a decrease or inactivation of lipocortin in the cell. Both the formation and the release of this substance are induced by In the absence of circulating steroids these glucocorticosteroids (4). effects will be impaired. After the addition of the Calcium-ionophore A23187, Calcium will enter the cell and lipocortin is phosphorylated (7). This effect will increase the activity of P1A2 and the release of the eicosanoids. The influence of derivatives on renal further research.
the increased formation of PC and lipoxygenase function in adrenalectomized rats is a subject of
REFERENCES 1.
Fanelli R. Castsgnoli MN, Salmona M. Chiabrando C, Noseda A, Characterization of arachidonic acid metabolic profiles in animal chromatography-mass spectrometry. tissues by high-resolution gas Biochem Biophys Acta 794: 292, 1984.
2.
Vincent JE, Zijlstra FJ. Comparison of the formation of prostaglandins in the kidneys of rats and rabbits. p.303 in Prostaglandin Synthetase Inhibitors: New Clinical Applications (P Ramwell, ed) Alan Lies, New York, 1980.
3.
Jim K. Hassid A, Sun F, Dunn M. Lipoxygenase activity in rat glomeruli, glomerular epithelial cells and cortical tubes. J Biol them 257: 10294, 1982.
4.
Blackwell GJ, Carnuccio R, DiRosa M, Flower RJ, Laugham CSJ. Parente L, Persico P. Russel-smith NC, Stone D. Glucocorticoids induce the formation and release of anti-inflammatory and anti-phospholipase proteins into the peritoneal cavity of the rat. Br J Pharmacol 76: 185, 1982.
5.
Salomon D. A Hirata F, Shiffmann E, Venkatasubramanian K. phospholipase A2 inhibitory protein in rabbit neutrophils induced by glucocorticoids. Proc Nat1 Acad Sci USA 77: 2533, 1980.
6.
Cloix JF, Colard 0, Rothhut B, Russo-Marie F. Characterization and partial purification of renocortins: two polypeptides formed in renal cells causing the anti-phospholipase-like action of glucocorticoids. Br J Pharmacol 79: 313, 1983.
7.
Hirata F, Matsuda K, Notsu Y, Hattori T, de1 Carmine R. Phosphorylation at a tyrosine residue of llpomodulin in mitogen-stimulated murine thymocytes. Proc Nat1 Acad Sci USA 81: 4717, 1984.
8.
Zijlstra FJ, Vincent JE. Determination of leukotrienes and prostaglandins in [14C] arachidonic acid labelled human lung tissue by high-performance liquid chromatography and radioimmunoassay. J Chromatogr 311: 39, 1984.
127
PR0ST.r
C
INCREASE IN THE FORMATION OF CYCLOOXYGENASE KIDNEYS OF ADRENALECTOMIZED RATS
AND
LIPOXYGENASE
PRODUCTS
IN
J.E. Vincent, F.J. Zijlstra and T.E. Gezel Department of Pharmacology, Faculty of Medicine Erasmus University Rotterdam, P.O.Box 1738 3000 DR Rotterdam, The Netherlands
ABSTRACT The effect of adrenalectomy on the formation of cyclooxygenase and lipoxygenase products from [1-14C]-arachidonic acid in rat kidneys after incubation with the Calcium-ibnophore A23187 has been determined. The metabolites were isolated by HPLC. The main components formed are PGD2, di-HETE and 5- ,12and 15-HETE. After PGE2. PGF2,, LTB4, 5 ,12 adrenalectomy, an increase occurs in the formation of PG and LT. which is highest in that of PGD2 and lP-HETE. These effects are most probably to a diminished formation or inactivation of lipocortin in related adrenalectomized animals, a glucocorticosteroid induced peptide with phospholipase A2 inhibitory activity. INTRODUCTION In rat kidneys, formation of metabolites of arachidonic acid 1) by both the cyclooxygenase and lipoxygenase pathways occurs. It has been reported that in this tissue PGD2, PGE2, PGF20, TxB2, 6-keto-PGF,o and 1 P-HETE are formed (1,2). In rat glomeruli, the main lipoxygenase product formed was 12-HETE (3). The initial reaction in the formation of these substances is the release of AA by the activation of P1A2. Recently, it has been shown that This in several types of cells an inhibitor of P1A2, lipocortin is present. substance has been isolated from macrophages, neutrophils and renomedullary interstitial formation is cells Its induced by (4,5,6). glucocorticosteroids. It has been demonstrated that in thymocytes, after lipocortin is inactivated by activation induced by several substances In this way, the inhibitor is no longer active and phosphorylation (7). P1A2 is activated, followed by the release of AA and the formation of metabolites. It is not known, whether the same mechanism exists in other 1)The following abbreviations were used: AA = arachidonic acid, LTs = leukotrienes, TxB2 = thromboxane B2, PGs = prostaglandins, 5- ,12- ,15- HETE = 5- ,12- ,15- hydroxy-6,8,11,14-eicosatetraenoic acid , P1A2 = diHETE = 5-.12-dihydroxy-6,8,11,14-eicosatetraenoic acid, phospholipase A2, adrex = adrenalectomized.
123
on the presence of is dependent cells. As the formation of lipocortin glucocorticosteroids, a decrease or inactivation may occur in adrex animals. In our experiments, the effect of adrenalectomy on the production of AA in rat kidneys after activation by the Calciumionophore A23187 metabolites was determined. MATERIALS AND METHODS were - Wistar strain male rats of 200g weight used. Animals was performed via a small medial incision. As drinking water Adrenalectomy a 1Z salt solution was given. Sham operated rats were used as controls, put The animals were used in the experiments several on normal drinking water. weeks after the operation. AA metobolite formation - The animals were sacrificed and the kidneys removed. The kidney weight was approx. lg. After chopping the tissue was incubated at 37'C and continuously gassed through a thin pipette with a mixture of 95% 02 and 5% COP. 2.5 uCi [1-14C] AA (55 mCi/mol) was added, luthathione (final cont. 2 mmol/l) and Ca-ionophore-A23187 (10 vmol/l), 4 H-LT and 'H-PG were added and the homogenate spun down (10 min, 14OOxg, respectively of 4OC). Serine and cysteine were added to prevent breakdown The pellets were washed once, and the LTC4 to LTD4 and LTD4 to LTE4. combined supernatants of each cell type were then applied to a couple of Sep-Pak C,8 and silica cartridges. The latter were prewashed with 10 ml ethanol and 10 ml distilled water. The samples were eluted with 5 ml ethanol on each column; these eluates were combined and evaporated to Thereafter, the dried dryness with a gentle stream of nitrogen at 37'C. in 0.5 ml solvent A (tetrahydrofuran-methanol-0.146 samples were dissolved EDTA in water-acetic acid, 25:30:45:0.1 to pH v/v, adjusted 5.5), centrifuged and purified by a Millex filter and kept in a HPLC microvial. Solvent system A was used for separation of lipoxygenase products on a Nucleosil 5C,8 column. The flow rate was 0.9 ml/min, the oven temperature 37Oc. Prostaglandins were separated on a Zorbax C8 column using system B (acetonitrile-benzene-water-acetic acid, 24:0.2:76:0.1 v/v), with a flow into the column and Volumes of 100 ,ul were injected rate of 2 ml/min. One minute chromatographed using a 1082B chromatograph (Hewlett Packard). The chromatographic fractions were collected for scintillation counting. method and the identification of the peaks have been described earlier (8).
PJateriols- Leukotriene B4 was a gift of Dr. J. Rokach (Merck Frosst A23187 was obtained from Hoechst, [1-14C] AA C ada Inc.). Ca-ionophore [Yn H(n)]5-HETE, ['H(n)] 12-HETE and C'H(nJ] 15-HETE were obtained from New ['H(n)] PGE2, [ H(n)]PGF20 and ['H(n)] PGD2 from England Nuclear and Sep-Pak C,8 and silica cartridges and HPLC filters, HA (0.45 urn), Amersham. FH (0.5 urn) and Millex (0.45 urn)were obtained from Waters/Millipore, HPLC microvials from Weidmann Plastics, Switzerland, Prepacked Nucleosil 5C18 and Zorbax C8 HPLC columns were obtained from Chrompack. RESULTS The substances formed from [1-14C] AA by the cyclooxygenase and lipoxygenase pathways in the kidneys after activation with the Ca-ionophore A23187 have been determined. In the control animals PGD2, POE2 and PGF2o were found. The substances LTB4, 5-HETE, 12-HETE and 15-HETE were present on the chromatogram as shown in fig. 1, representing a separation of lipoxygenase products. 124
(5S,12S)-5,12-dihydroxy-6,8,10 trons-14 identified as One peak was cis-eicosatetraenoic acid (M. Claeys, personal communication). The amounts The differences formed in .the sham operated rats are shown in table I. between the amounts formed by the control and adrex rats are represented in fig. 2. There were large increases in the formation of PGD2 and 12-HETE and smaller ones in the other products. 4000
3000
w f
2000
5 P
1000
10
20
30
40
Time
50
60
70
80
(min)
Fig. 1. Chromatogram of lipoxygenase products formed from [1-'4C] AA by chopped kidney tissue of a sham operated rat after stimulation with A23187.
Table
I.
Formation of eicosanoids from operated rats. (n=5)
[1-'4C] AA
dpm x 1000/g
gg2a
10.5 6.1
LTB2 PGD2
<0.2 5.7 f ?? 0.1 1.4
5 -i&c* 12-HETE 15-HETE
28.1 f 4.7 31.4 f 8.2 16.8 f 2.5
i
2.4 1.5
125
in
kidneys of
sham
I PGF2,
-
PGE2
T*
T*
1
L
-
PCD, HETE
HETE
A 5-
HETE
Fig. 2. Difference in formation of cyclooxygenase and lipoxygenase products formed from [l-14C] AA in kidneys of sham operated and adrenalectomized rats after activation by A23187. Data f SEM. *P
DISCUSSION of the In the kidney. the medulla is the main site of production These substances have been implicated in several aspects of prostaglandins. kidney function such as the regulation of renal blood flow, water and sodium filtration. PGD2 induces glomerular renin transport, release and It may prevent the aggregation. vasodilatation and inhibits platelet formation of platelet aggregates which can impair glomerular filtration. The role of the lipoxygenase derivatives in the kidney is not well known. The effects of LTB4 are chemotaxis and the induction of increased vascular The 5- and 12-HETEs also have small chemotactic effects. permeability. mineral0 After adrenalectomy, the secretion of glucocorticosteroids, corticosteroids and adrenaline will rapidly diminish and disappear. The absence of circulating steroids will lead to an increase in the secretion of How does this affect the metabolism of unsaturated fatty acids ? No ACTH. action of mineralocorticosteroids has yet been demonstrated on this factor. and ACTH both induce the lipolysis of adipose tissue, leading to Adrenaline the release of fatty acids. This effect is very small in the absence of glucocorticosteroids.
126
As has been mentioned before, the effects of adrenalectomy can mOSt probably be ascribed to a decrease or inactivation of lipocortin in the cell. Both the formation and the release of this substance are induced by In the absence of circulating steroids these glucocorticosteroids (4). effects will be impaired. After the addition of the Calcium-ionophore A23187, Calcium will enter the cell and lipocortin is phosphorylated (7). This effect will increase the activity of P1A2 and the release of the eicosanoids. The influence of derivatives on renal further research.
the increased formation of PC and lipoxygenase function in adrenalectomized rats is a subject of
REFERENCES 1.
Fanelli R. Castsgnoli MN, Salmona M. Chiabrando C, Noseda A, Characterization of arachidonic acid metabolic profiles in animal chromatography-mass spectrometry. tissues by high-resolution gas Biochem Biophys Acta 794: 292, 1984.
2.
Vincent JE, Zijlstra FJ. Comparison of the formation of prostaglandins in the kidneys of rats and rabbits. p.303 in Prostaglandin Synthetase Inhibitors: New Clinical Applications (P Ramwell, ed) Alan Lies, New York, 1980.
3.
Jim K. Hassid A, Sun F, Dunn M. Lipoxygenase activity in rat glomeruli, glomerular epithelial cells and cortical tubes. J Biol them 257: 10294, 1982.
4.
Blackwell GJ, Carnuccio R, DiRosa M, Flower RJ, Laugham CSJ. Parente L, Persico P. Russel-smith NC, Stone D. Glucocorticoids induce the formation and release of anti-inflammatory and anti-phospholipase proteins into the peritoneal cavity of the rat. Br J Pharmacol 76: 185, 1982.
5.
Salomon D. A Hirata F, Shiffmann E, Venkatasubramanian K. phospholipase A2 inhibitory protein in rabbit neutrophils induced by glucocorticoids. Proc Nat1 Acad Sci USA 77: 2533, 1980.
6.
Cloix JF, Colard 0, Rothhut B, Russo-Marie F. Characterization and partial purification of renocortins: two polypeptides formed in renal cells causing the anti-phospholipase-like action of glucocorticoids. Br J Pharmacol 79: 313, 1983.
7.
Hirata F, Matsuda K, Notsu Y, Hattori T, de1 Carmine R. Phosphorylation at a tyrosine residue of llpomodulin in mitogen-stimulated murine thymocytes. Proc Nat1 Acad Sci USA 81: 4717, 1984.
8.
Zijlstra FJ, Vincent JE. Determination of leukotrienes and prostaglandins in [14C] arachidonic acid labelled human lung tissue by high-performance liquid chromatography and radioimmunoassay. J Chromatogr 311: 39, 1984.
127
PR0ST.r
C