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Changes in prostacyclin and thromboxane biosynthesis and their catabolic enzyme activity in kidneys of aging rats
Life Sciences, Vol. 34, pp. 1269-1280 Printed in the U.S.A.
Pergamon Press
CHANGES IN PROSTACYCLIN AND THROMBOXANE BIOSYNTHESIS AND THEIR CATABOLIC ENZYME ACTIVITY IN KIDNEYS OF AGING RATS
Wen-Chang Chang and Hstn-Hstung Tai Division of Medicinal Chemistry and Pharmacognosy College of Pharmacy, University of Kentucky Lexington, KY 40536-0053, USA (Received in final form January 17, 1984)
Surmar 7 The effects of aging on the prostacyclin and thromboxane biosynthesis and prostaglandin catabolic enzyme activity in rat kidney were investigated. The prostacyclin biosynthesis, using arachidonic acid as substrate, was the greatest in young kidneys (2 months old) and then progressively decreased in mature (12 months old) and old (24 months old) kidneys, while thromboxane biosynthetic activity showed no significant change as a function of age. When prostaglandin H^ was used as substrate, the prostacyclin and thromboxan~ biosynthesis showed similar results as when arachidonic acid was used as substrate; the prostacyclin biosynthesis progressively decreased and thromboxane biosynthesis showed no significant change as a function of age. The fatty acid cyclooxygenase in kidney was measured by a specific radioimmunoassay. No significant change in renal fatty acid cyclooxygenase as a function of age was found. Thus, we concluded that the progressive decrease in renal prostacyclin biosynthesis as a function of age is due to a defect in prostacyclin synthetase in + aged kidneys. The prostaglandin catabolic enzyme, NAD -dependent 15-hydroxyprostaglandin dehydrogenase, in kidneys was also investigated. The enzyme activity progressively decreased as a function of age, which suggested a decrease in the metabolism of thromboxane A^ in aged kidneys. The present results, inz dicating a decrease in renal prostacyclin biosynthesis and renal 15-hydroxyprostaglandin dehydrogenase activity with aging, might contribute to a plausible explanation of the progressive decrease in renal functions in the elderly. Recently it has been suggested that a balance between prostacyclin and thromboxane A_ plays an important role in vascular homeostasis and thrombus formation (11~. In the vascular system, we have reported that prostacyclin production by vascular cells and vascular walls decreases with aging, and have suggested that the decrease in prostacyclin formation by vascular walls may partially account for the pathogenesis of spontaneous atherosclerosis and arterial thrombus in the elderly (2,4).
0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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Aging and Renal Arachidonate Metabolism
Vol. 34, No. 13, 1984
The kidney is responsible for the elimination of most of the nonvolatile waste products of metabolism from the body. The renal functions, including the renal blood flow and glomerular filtration rate, progressively decrease with aging (9). The rate at which glomerular filtrate is formed is dependent primarily upon the rate of blood flow through the kidney. Therefore, the regulation of renal blood flow seems to be very important in the renal functions. Prostacyclin and thromboxane are two of the major products transformed from arachidonic acid by rat kidney (21). Since prostacyclln is a potent vasodilator and thromboxane A 2 a potent vasoconstrictor, the change in their biosynthesis in the kidney may be important in the determination of renal blood flow. In this study, we investigated the changes in prostacyclin and thromboxane biosynthesis and prostaglandin catabolic activity as a function of age, and suggested that the changes in prostacyclin and thromboxane biosynthesis in kidneys might contribute to the decrease in the renal function in the aged.
Materials and Methods Chemicals Arachidonlc acid, human hemoglobin (type IV), d,l-eplnephrine, bovine liver glutamic dehydrogenase (50 Ulmg), nicotinamide adenine dinucleotide (NAD+), a-ketoglutarate monosodium salt and bovine serum albumin (fraction V) were purchased from Sigma Chemical Co. Prostaglandlns and thromboxane B 2 standards were generous gifts of The Upjohn Co. and Ono Pharmaceutical Co., Ltd., Tokyo, Japan. All reagents not specified above were of analytical
grade. Animals Male, Virgin COBS R CDF R F344/Crl rats aged 2 (young), 12 (mature) and 24 (old) months, bred by The Charles River Breeding Laboratories, Inc., Wilmington, MA, were kindly SUpl~lled by the National Institute on Aging, NIH. Upon receipt, all animals were maintained on a commercial laboratory feed and water was provided ad libltum. Preoaration of microsomal and c~tosolic fractions of kidney Animals were sacrificed by bleeding under the anesthesia of diethyl ether and the kidneys were rapidly removed. Kidneys were homogenized in 50 mM TrisHCI buffer, pH 7.4 (tissue to buffer ratio of 1:4, w/v) using a Teflon homogenizer. The homogenate was centrifuged at 9,000 xg for 20 min, and the resuiting supernatant was recentrifuged at 105,000 xg for 1 h by a Beckman Model L5-50 ultracentrifuge. The resulting supernatants were designated as the cytosolic fraction. The pellets were resuspended in 50 mM Tris-HCl buffer (pH 7.4) and designated as the microsomal fraction. Preparation of prosta~landin H 2 Prostaglandin H^ was enzymatically synthesized from arachidonic acid z with the microsomal fraction of sheep seminal vesicles according to the methods described by Hamberg et al. (6).
Vol. 34, No. 13, 1984
Aging and Renal Arachidonate Metabolism
Prosta~landin biosynthetic
activities
1271
in kidneys
Each assay tube contained: hemoglobin, I B M ; epinephrine, 1 mM; arachidonic acid, I0 pM; and appropriate amount of microsomal fraction in I ml of 50 mM Tris-HCI buffer, pH 7.4. Incubation was carried out at 37°C for 30 min. The reaction was terminated by acidification to pH 3.0. The pH of the reaction mixture was then reneutralized, and the formation of 6-ketoprostaglandin FI~ and thromboxane B 2 was determined by the respective radioimmunoassays. In the experiments using prostaglandin H 2 as a substrate, each assay tube contained 14.3 ~M prostaglandin H~ and appropriate amount of microsomal fraction in 0.5 ml of 50 mM Tris-HCl buffer, pH 7.4. The reaction was allowed to proceed at room temperature for 2 min. Termination of the reaction and preparation of samples for the determination of 6-ketoprostaglandin FI= and thromboxane B 2 production were performed as mentioned above. Radioimmunoassays
of 6-ketoprosta~landin
F1
and thromboxane
Specific radioimmunoassays for 6-ketoprostaglandin F. B^ immunoreactivity were performed according to the m e t h o ~ ously (16,19). Radioimmunoassay
of fatty acid cyclooxygenase
B2 and thromboxane described previ-
in kidney
The microsomal fractions of kidneys were solubilized in an aliquot of standard radioimmunoassay buffer (50 mM Tris-HCl, pH 7.4, O.1% gelatin) containing 1% Tween-20 and diluted with the radioimmunoassay buffer containing 0.1% Tween-20 for the use of radioimmunoassay. Radioimmunoassay of fatty acid cyclooxygenase was performed as previously described (18,3). The antiserum was produced in rabbits by repeated immunization with the purified sheep seminal vesicle enzyme (15). Cyclooxygenase was labeled by ~chloramine T mediated radioiodination and was purified by Sephadex G-IO and hydroxyapatite chromatography in the presence of O.I~ Tween-20. Antiserum, at final dilution of 1:3750, was used for the assay. Separation of bound from free enzyme was achieved by the double antibody method. The properties of antiserum and the methods of radioimmunoassay, used in the present studies, were almost the same as those reported recently by Roth and Machuga (14). 15-Hydroxyprosta~landin
dehydro~enase
assay
3 Preparation of the substrate (15S) ~5- H~prostaglandin E? and the assay were performed as previously described (17). In brief, the incubation mixture contained~ NHLCI (5 Bmol), monosodium =-ketoglutarate (i ~mol), NAD + (i ~mol), (15S)-~15-O~-p~ostaglandin E 2 (I nmol, 20,000 cpm), glutamate dehydrogenase (iO0 ~g), and appropriate amounts of kidney cytosol in a final volume of 1 ml of 50 mM Tris-HCl buffer, pH 7.4. The reaction mixture was incubated for 10 min at 37°C and terminated by the addition of 0.3 ml of a charcoal suspension (10% in water). The reaction mixture was centrifuged at i,OOO xg for 5 min after standing for 5 min at room temperature. The supernatant was decanted and the radioactivity was determined by liquid scintillation counting.
Protein determinations Protein contents were determined by the method of Lowry et al. (i0) with bovine serum albumin (fraction V) as a standard.
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Aging and Renal Arachidonate Metabolism
Vol. 34, No. 13, 1984
Results Effect of aging on the conversion of arachidonic
acid by kidney microsomes
Conversion of arachidonic acid to prostacyclin and thromboxane A_ by kidney microsomes was determined by the formation of their stable, hydroi~zed metabolites, 6-ketoprostaglandin F. and thromboxane B., respectively. The IQ K biosynthesis of 6-ketoprostaglandin F. and thromboxane B_ was measured by ~. specific radioimmunoassays. Our prellmlnary experiments s~owed that their formation is nearly linear up to 30 min of incubation. The effects of aging on the conversion of arachidonic acid to 6-ketoprostaglandin Fla and thromboxane B2 by kidney microsomes are shown in Figs. 1 and 2. A progressive decrease in prostacyclin biosynthesis in kidney microsomes was observed. Both of the decreases, from young to mature animals, and from mature to old animals, were statistically significant ( P ~ 0.O1). On the other hand, no significant change in thromboxane biosynthesis was found.
,o) 10
9 [
8
7
6
c
(lO)
s
5
4 ,El-
3
!
I
~o
1
Young
Mature
(10)
Old
FIG. I Effect of age on the biosynthesis of prostacyclin from arachidonic acid in kidneys. Values represent the mean + S.E.M. for the number of animals indicated in parenethses. 6-Keto PGFIa is 6-ketoprostaglandin F1 .
Vol. 34, No. 13, 1984
Aging and Renal Arachidonate Metabolism
1273
t~0)
10 9 8 Win.
I:
7
m
0 0k ,
qP
6
Q.
5
m
E D
4
C
3
m X
2 1 0
Young
Mature
Old
FIG. 2 Effect of age on the biosynthesis of thromboxane from arachidonic acid in kidneys. Values represent the mean + S.E.M. for the number of animals indicated in parentheses. TXB 2 is thromboxane B 2.
Effect of a6in ~ on the conversion of prosta~landin H 2 by kidney microsomes The preliminary experiments showed that the formation of 6-ketoprostaglandin F . and thromboxane B. is nearly linear during the first 2 min of 2 incubation~'~unde r our assay conditions and this time range was therefore chosen. The effects of aging on the conversion of prostaglandin H to 6-ketoprostaglandin F. and thromboxane B^ are shown in Figs. 3 and 42 A progres• ~ . L . . slve decrease in prostacyclin blosynthesls from prostaglandln H 2 by kidney microsomes was observed. Both of the decreases, from young to mature and from mature to old animals, were statistically significant. However, no significant change in thromboxane biosynthesis from prostaglandin H 2 in kidney microsomes was observed. Effect of asing on the content of fatty acid cyclooxy~enase in kidney The homogeneous sheep seminal vesicular cyclooxygenase was used as standards for the radioimmunoassay of fatty acid cyclooxygenase. The inhibition curve has been reported to be steepest in the range of 5 to 50 ng of standard (3). Therefore, the assays for the samples in the present studies
1274
Aging and Renal Arachidonate Metabolism
120 "
"~
100
o O.
80
Vol. 34, No. 13, 1984
(10)
I
m
E C
60
(10)
II
u_
0
eL
II 40
0
(10)
o I
~0
II
t!
20
0
Young
Mature FIG.
Old
3
Effect of age on the biosynthesis of prostacyclin from prostaglandin H_ in kidneys. Values represent the mean + S.E.M. for the number of animals i~dicared in parentheses. 6-Keto PGFI~ is 6-ketoprostaglandin Fld.
Vol. 34, No. 13, 1984
Aging and Renal Arachidonate Metabolism
1275
140(10) 120
(lO) (~o)
100
t
8O
I
mE
CI
E
60
D
C
c~
40
i
X
I-
20
O"
i
Young
Mature
Old
FIG. 4
Effect of age on the biosynthesis of thromboxane from prostaglandin H^ in kidneys. Values represent the mean + S.E.M. for the number of animals i~dlcared in parentheses. TXB 2 is thromb~xane B 2.
1276
Aging and Renal Arachidonate Metabolism
were performed in this range. acid cyclooxygenase in kidneys observed.
Vol. 34, No. 13, 1984
The effect of aging on the content of fatty is shown in Fig. 5. No significant change was
20
(lO) 18
(lO)
(lO)
÷
16
--t-
14
'°
6 4 2 O
Young
Mature
Old
FIG. 5 Effect of age on the content of fatty acid cyclooxygenase in kidneys. Values represent the mean + S.E.M. for the number of animals indicated in parentheses.
Effect of a~in~ on 15-hydroxyprostaslandin
dehydro~enase
activity in kidney
Fig. 6 shows the effect of aging on 15-hydroxyprostaglandin dehydrogenase activity in kidney cytosol. Kidney cytosol of young rats showed the highest enzyme activity. A progressive decrease as a function of age was found. Both of the decreases in the enzyme activity from young to mature animals, and from mature to old animals, were statistically significant (PCO.OI).
Vol. 34, No. 13, 1984
Aging and Renal Arachidonate Metabolism
1277
6 -
(lO) ~0~
5
X g --
4
ot _ a_ m
3
E E a_ "D
{10)
I
2
4'1 m
) 4u
m
1
<
(10)
o i
Young
Mature
Old
FIG. 6 Effect of age on NAD+-dependent 15-hydroxyprostaglandin dehydrogenase activity in kidneys. Values represent the mean + S.E.M. for the number of animals indicated in parentheses.
Discussion Incubation of cortical and medullary microsomal preparation of rat kidneys with arachidonic acid and prostaglandin H_ shows that both the cortex and medulla could synthesize prostacyclin and t~romboxane (21). Both prostacyclin and thromboxane are also the major products transformed from arachidonic acid by the microsomal preparation of human kidneys, and the biosynthesis of prostacyclin is more prominent than that of prostaglandin E p in human kidneys (7). The thromboxane biosynthesis is of renal parenchymal origin, since the presence of significant thromboxane biosynthesis by cultured glomerular epithelial cells is observed (13). Therefore, the balance between prostacyclin and thromboxane A 2 biosynthesis in kidneys might play an important role in the regulation of renal blood flow, since prostacyclin is a
1278
Aging and Renal Arachidonate Metabolism
Vol. 34, No. 13, 1984
potent vasodilator and thromboxane Ap a potent vasoconstrictor. In fact, a significant role of renal prostaglanains and thromboxane in the regulation of renal blood flow has been suggested. Blasingham et al. (i) reported that the administration of cyclooxygenase inhlbitors results in decrease in renal blood flow. Morrison et al. (12) have observed that thromboxane A 2 is unmasked in ureterally obstructed perfused kidneys, and thromboxane A 2 might mediate the postobstructive vasoconstriction. In the present investigation, we studied the changes in prostacyclin and thromboxane biosynthetic activities in the microsomal fraction of rat kidneys as a function of age. Conversion of arachidonic acid to prostacyclin progressively decreases with aging (Fig. I), while the formation of thromboxane from arachidonic acid does not significantly change (Fig. 2). In the metabolism of arachidonic acid, it is first converted to prostaglandin endoperoxides (G2, H 2) by fatty acid cyclooxygenase, and then to prostacyclln by prostacyc[in synthetase and to thromboxane A 2 by thromboxane synthetase. If the decrease in prostacyclin biosynthesis in rat kidney with aging is due to a defect in fatty acid cyclooxygenase, it is reasonable to expect that a decrease in thromboxane synthesis should also be observed. In fact, we only observed the decrease in prostacyclin biosynthesis, not in thromboxane biosynthesis. These results indicate that the decrease in the renal prostacyclin biosynthesis in old animals might be due to a defect in prostacyclln synthetase. In order to check this possibility, prostaglandin H 2 was used as substrate. The conversion of prostaglandin H 2 to prostacyclin by kidney microsomes decreases as a function of age (Fig. 3), while the transformation of prostaglandin H. to thromboxane does not show any significant change (Fig. 4). Moreover, t~e immunoreactive fatty acid cyclooxygenase in kidney microsomes does not show any significant change as a function of age (Fig. 5). We therefore concluded that the decrease in prostacyclin biosynthesis in rat kidney with aging is due to a defect in prostacyclin synthetase, not in fatty acid cyclooxygena~e. The present results in kidneys are consistent with our previous results showing an age-related decrease in prostacyclin synthetase activity in cultured rat aortic smooth muscle cells isolated from mature and old rats (2). NAD+-dependent 15-hydroxyprostaglandin dehydrogenase is the first enzyme to metabolize prostaglandins. Kidney is one of the tissues abundant with this enzyme. Prostacyclin is metabolized by renal 15-hydroxyprostaglandin dehydrogenase as efficiently as prostaglandin E[ and F 2 (20). In the metabolism of thromboxane A_ there is no direct evldence showing the thromboxane A_ metabolism by l~-hydroxyprostaglandin dehydrogenase because the standarl thromboxane A^ is not yet available. However, two results to date strongly indicate that thromboxane A ~ is metabolized by 15-hydroxyprostaglandin dehydrogenase. Dawson et al. ('5) analyzed the metabolites of arachidonic acid in guinea pig lung after immunological challenge, and detected more 15-ketothromboxane B_ than thromboxane B_ itself in the arachidonate metabolites. Kung-chao and Tai (8) found that t~romboxane Bp is a very poor substrate for renal 15-hydroxyprostaglandin dehydrogenase. Therefore, it is reasonable to assume that thromboxane A^ is first metabolized by 15-hydroxyprostaglandin dehydrogenase to 15-keto-t~romboxane A 2 and then non-enzymatically converted to 15-keto-thromboxane B^. Therefore, the change in renal 15-hydroxyprostaglandin dehydrogenase activity with aging might have some implications in the physiological function of kidney. The enzyme activity is the highest in young animals (Fig. 6). Since the renal prostacyclin and thromboxane biosynthetic activities are relatively high in young animals, both the prostacyclin
Vol. 34, No. 13, 1984
Aging and Renal Arachidonate Metabolism
1279
and thromboxane A 2 biosynthesized might be equally metabolized by 15-hydroxyprostaglandin deKydrogenase. Consequently, the ratio of renal prostacyclin/ thromboxane A 2 might remain unchanged in young animals. In old animals, the renal prostacyclin biosynthesis is very low, while the renal thromboxane biosynthesis is as active as that in young animals. The decrease in the renal 15-hydroxyprostaglandin dehydrogenase activity might prolong the vasoconstrictive effect of thromboxane A 2. Consequently, it might slow the renal blood flow in old animals. In summary, the present results, indicating the decrease in renal prostacyclin biosynthesis and renal 15-hydroxyprostaglandin dehydrogenase activity with aging, might contribute a plausible explanation of progressive decreases of renal functions in the elderly. Further investigation on the direct involvement of prostacyclin and thromboxane A 2 in the renal functions in vivo in different ages of animals is needed.
Acknowledsements The authors wish to thank the National Institute on Aging, NIH for the supply of animals and to Mrs. Sharon Parker for her excellent technical assistance. Thanks are also due to Mr. Rong-Fong Shen for his assistance in the preparation of prostaglandin H2, and to The Upjohn Co., Kalamazoo, MI, U.S.A. and Ono Pharmaceutical Co., Osaka, Japan for the supply of prostaglandin and thromboxane B 2 standards. We are indebted to MrSw~s. West for her assistance in the preparation of the manuscript. This work supported in part from the American Heart Association Kentucky Affiliate, Inc., the University of Kentucky Isabell L. Kircher Fund, the National Institutes of Health (GM 30380) and the American Heart Association (81-1152).
References I. 2. 3. 4. 5. 6. 7. 8. 9.
i0. II. 12.
M. C. BLASINGHAM, R. E. SHADE, L. SHARE and A. NASJLETTI, J. Pharmacol. Exp. Ther. 214 i-4 (1980). W. C. CHANG, S. MUROTA, J. NAKAO and H. ORIMO, Biochim. Biophys. Acta 620 159-166 (1980). W. C. CHANG, J. NAKAO, S. MUROTA and H. H. TAI, Prostaglandins Leukotrienes Med. I0 33-37 (1983). W. C. CHANG and H. H. TAI, Prostaglandins Leukotrienes Med. (in press). W. DAWSON, J. R. BOOT, A. F. COCKERILL, D. N. B. MALLEN and D. J. OSBORNE, Nature 262 699-702 (1976). M. HAMBERG, J. SVENSSON, T. WAKABAYASHI and B. SAMUELSSON, Proc. Natl. Acad. Sci. USA 71 345-349 (1974). A. HASSID and M. J. DUNN, J. Biol. Chem. 255 2472-2475 (1980). D. T. Y. KUNG-CHAO and H. H. TAI, Biochim. Biophys. Acta 614 1-13 (1980) R. D. LINDEMAN, In The Physiology and Pathology of Human Aging (R. Goldman and M. Rockstein, ed.) pp. 19-38, Academic Press Inc., New York, San Francisco, London (1975). O. H. LOWRY, N. J. ROSENBROUGH, A. L. FARR and R. J. RANDALL, J. Biol. Chem. 193 265-275 (1951). S. MONCADA and J. R. VANE, Pharmacol. Rev. 30 293-331 (1979). A. R. MORRISON, K. NISHIKAWA and P. NEEDLEMAN, J. Pharmacol. Exp. Ther. 205 i-8 (1978).
1280
13. 14. 15. 16. 17. 18. 19. 20. 21.
Aging and Renal Arachidonate Metabolism
Vol. 34, No. 13, 1984
A. PETRULIS, M. AIKAWA and M. J. DUNN, Kidney Int. 20 469-474 (1981). G. J. ROTH and E. T. MACHUGA, J. Lab. Clin. Med. 99 187-196 (1982). W. L. SMITH and G. P. WILKIN, Prostaglandins 1 3 873-892 (1977). C. L. TAI and H. H. TAI, Prostaglandins Med. 4 399-408 (1980). H. H. TAI, Biochemistry 15 4586-4592 (1976). H. H. TAI, C. L. TAI and W. L. SMITH, Fed. Proc. 39 323 (1980). H. H. TAI and B. YUAN, Anal. Biochem. 87 343-349 ('[978). P. Y. K. WONG, J. C. McGIFF, L. CAGEN, K. U. MALIK and F. F. SUN, J. Biol. Chem. 254 12-14 (1979). T. V. ZENSER, C. A. HERMAN, R. R. GORMAN and B. B. DAVIS, Biochem. Biophys. Res. Commun. 79 357-363 (1977).
Pergamon Press
CHANGES IN PROSTACYCLIN AND THROMBOXANE BIOSYNTHESIS AND THEIR CATABOLIC ENZYME ACTIVITY IN KIDNEYS OF AGING RATS
Wen-Chang Chang and Hstn-Hstung Tai Division of Medicinal Chemistry and Pharmacognosy College of Pharmacy, University of Kentucky Lexington, KY 40536-0053, USA (Received in final form January 17, 1984)
Surmar 7 The effects of aging on the prostacyclin and thromboxane biosynthesis and prostaglandin catabolic enzyme activity in rat kidney were investigated. The prostacyclin biosynthesis, using arachidonic acid as substrate, was the greatest in young kidneys (2 months old) and then progressively decreased in mature (12 months old) and old (24 months old) kidneys, while thromboxane biosynthetic activity showed no significant change as a function of age. When prostaglandin H^ was used as substrate, the prostacyclin and thromboxan~ biosynthesis showed similar results as when arachidonic acid was used as substrate; the prostacyclin biosynthesis progressively decreased and thromboxane biosynthesis showed no significant change as a function of age. The fatty acid cyclooxygenase in kidney was measured by a specific radioimmunoassay. No significant change in renal fatty acid cyclooxygenase as a function of age was found. Thus, we concluded that the progressive decrease in renal prostacyclin biosynthesis as a function of age is due to a defect in prostacyclin synthetase in + aged kidneys. The prostaglandin catabolic enzyme, NAD -dependent 15-hydroxyprostaglandin dehydrogenase, in kidneys was also investigated. The enzyme activity progressively decreased as a function of age, which suggested a decrease in the metabolism of thromboxane A^ in aged kidneys. The present results, inz dicating a decrease in renal prostacyclin biosynthesis and renal 15-hydroxyprostaglandin dehydrogenase activity with aging, might contribute to a plausible explanation of the progressive decrease in renal functions in the elderly. Recently it has been suggested that a balance between prostacyclin and thromboxane A_ plays an important role in vascular homeostasis and thrombus formation (11~. In the vascular system, we have reported that prostacyclin production by vascular cells and vascular walls decreases with aging, and have suggested that the decrease in prostacyclin formation by vascular walls may partially account for the pathogenesis of spontaneous atherosclerosis and arterial thrombus in the elderly (2,4).
0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
1270
Aging and Renal Arachidonate Metabolism
Vol. 34, No. 13, 1984
The kidney is responsible for the elimination of most of the nonvolatile waste products of metabolism from the body. The renal functions, including the renal blood flow and glomerular filtration rate, progressively decrease with aging (9). The rate at which glomerular filtrate is formed is dependent primarily upon the rate of blood flow through the kidney. Therefore, the regulation of renal blood flow seems to be very important in the renal functions. Prostacyclin and thromboxane are two of the major products transformed from arachidonic acid by rat kidney (21). Since prostacyclln is a potent vasodilator and thromboxane A 2 a potent vasoconstrictor, the change in their biosynthesis in the kidney may be important in the determination of renal blood flow. In this study, we investigated the changes in prostacyclin and thromboxane biosynthesis and prostaglandin catabolic activity as a function of age, and suggested that the changes in prostacyclin and thromboxane biosynthesis in kidneys might contribute to the decrease in the renal function in the aged.
Materials and Methods Chemicals Arachidonlc acid, human hemoglobin (type IV), d,l-eplnephrine, bovine liver glutamic dehydrogenase (50 Ulmg), nicotinamide adenine dinucleotide (NAD+), a-ketoglutarate monosodium salt and bovine serum albumin (fraction V) were purchased from Sigma Chemical Co. Prostaglandlns and thromboxane B 2 standards were generous gifts of The Upjohn Co. and Ono Pharmaceutical Co., Ltd., Tokyo, Japan. All reagents not specified above were of analytical
grade. Animals Male, Virgin COBS R CDF R F344/Crl rats aged 2 (young), 12 (mature) and 24 (old) months, bred by The Charles River Breeding Laboratories, Inc., Wilmington, MA, were kindly SUpl~lled by the National Institute on Aging, NIH. Upon receipt, all animals were maintained on a commercial laboratory feed and water was provided ad libltum. Preoaration of microsomal and c~tosolic fractions of kidney Animals were sacrificed by bleeding under the anesthesia of diethyl ether and the kidneys were rapidly removed. Kidneys were homogenized in 50 mM TrisHCI buffer, pH 7.4 (tissue to buffer ratio of 1:4, w/v) using a Teflon homogenizer. The homogenate was centrifuged at 9,000 xg for 20 min, and the resuiting supernatant was recentrifuged at 105,000 xg for 1 h by a Beckman Model L5-50 ultracentrifuge. The resulting supernatants were designated as the cytosolic fraction. The pellets were resuspended in 50 mM Tris-HCl buffer (pH 7.4) and designated as the microsomal fraction. Preparation of prosta~landin H 2 Prostaglandin H^ was enzymatically synthesized from arachidonic acid z with the microsomal fraction of sheep seminal vesicles according to the methods described by Hamberg et al. (6).
Vol. 34, No. 13, 1984
Aging and Renal Arachidonate Metabolism
Prosta~landin biosynthetic
activities
1271
in kidneys
Each assay tube contained: hemoglobin, I B M ; epinephrine, 1 mM; arachidonic acid, I0 pM; and appropriate amount of microsomal fraction in I ml of 50 mM Tris-HCI buffer, pH 7.4. Incubation was carried out at 37°C for 30 min. The reaction was terminated by acidification to pH 3.0. The pH of the reaction mixture was then reneutralized, and the formation of 6-ketoprostaglandin FI~ and thromboxane B 2 was determined by the respective radioimmunoassays. In the experiments using prostaglandin H 2 as a substrate, each assay tube contained 14.3 ~M prostaglandin H~ and appropriate amount of microsomal fraction in 0.5 ml of 50 mM Tris-HCl buffer, pH 7.4. The reaction was allowed to proceed at room temperature for 2 min. Termination of the reaction and preparation of samples for the determination of 6-ketoprostaglandin FI= and thromboxane B 2 production were performed as mentioned above. Radioimmunoassays
of 6-ketoprosta~landin
F1
and thromboxane
Specific radioimmunoassays for 6-ketoprostaglandin F. B^ immunoreactivity were performed according to the m e t h o ~ ously (16,19). Radioimmunoassay
of fatty acid cyclooxygenase
B2 and thromboxane described previ-
in kidney
The microsomal fractions of kidneys were solubilized in an aliquot of standard radioimmunoassay buffer (50 mM Tris-HCl, pH 7.4, O.1% gelatin) containing 1% Tween-20 and diluted with the radioimmunoassay buffer containing 0.1% Tween-20 for the use of radioimmunoassay. Radioimmunoassay of fatty acid cyclooxygenase was performed as previously described (18,3). The antiserum was produced in rabbits by repeated immunization with the purified sheep seminal vesicle enzyme (15). Cyclooxygenase was labeled by ~chloramine T mediated radioiodination and was purified by Sephadex G-IO and hydroxyapatite chromatography in the presence of O.I~ Tween-20. Antiserum, at final dilution of 1:3750, was used for the assay. Separation of bound from free enzyme was achieved by the double antibody method. The properties of antiserum and the methods of radioimmunoassay, used in the present studies, were almost the same as those reported recently by Roth and Machuga (14). 15-Hydroxyprosta~landin
dehydro~enase
assay
3 Preparation of the substrate (15S) ~5- H~prostaglandin E? and the assay were performed as previously described (17). In brief, the incubation mixture contained~ NHLCI (5 Bmol), monosodium =-ketoglutarate (i ~mol), NAD + (i ~mol), (15S)-~15-O~-p~ostaglandin E 2 (I nmol, 20,000 cpm), glutamate dehydrogenase (iO0 ~g), and appropriate amounts of kidney cytosol in a final volume of 1 ml of 50 mM Tris-HCl buffer, pH 7.4. The reaction mixture was incubated for 10 min at 37°C and terminated by the addition of 0.3 ml of a charcoal suspension (10% in water). The reaction mixture was centrifuged at i,OOO xg for 5 min after standing for 5 min at room temperature. The supernatant was decanted and the radioactivity was determined by liquid scintillation counting.
Protein determinations Protein contents were determined by the method of Lowry et al. (i0) with bovine serum albumin (fraction V) as a standard.
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Aging and Renal Arachidonate Metabolism
Vol. 34, No. 13, 1984
Results Effect of aging on the conversion of arachidonic
acid by kidney microsomes
Conversion of arachidonic acid to prostacyclin and thromboxane A_ by kidney microsomes was determined by the formation of their stable, hydroi~zed metabolites, 6-ketoprostaglandin F. and thromboxane B., respectively. The IQ K biosynthesis of 6-ketoprostaglandin F. and thromboxane B_ was measured by ~. specific radioimmunoassays. Our prellmlnary experiments s~owed that their formation is nearly linear up to 30 min of incubation. The effects of aging on the conversion of arachidonic acid to 6-ketoprostaglandin Fla and thromboxane B2 by kidney microsomes are shown in Figs. 1 and 2. A progressive decrease in prostacyclin biosynthesis in kidney microsomes was observed. Both of the decreases, from young to mature animals, and from mature to old animals, were statistically significant ( P ~ 0.O1). On the other hand, no significant change in thromboxane biosynthesis was found.
,o) 10
9 [
8
7
6
c
(lO)
s
5
4 ,El-
3
!
I
~o
1
Young
Mature
(10)
Old
FIG. I Effect of age on the biosynthesis of prostacyclin from arachidonic acid in kidneys. Values represent the mean + S.E.M. for the number of animals indicated in parenethses. 6-Keto PGFIa is 6-ketoprostaglandin F1 .
Vol. 34, No. 13, 1984
Aging and Renal Arachidonate Metabolism
1273
t~0)
10 9 8 Win.
I:
7
m
0 0k ,
qP
6
Q.
5
m
E D
4
C
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m X
2 1 0
Young
Mature
Old
FIG. 2 Effect of age on the biosynthesis of thromboxane from arachidonic acid in kidneys. Values represent the mean + S.E.M. for the number of animals indicated in parentheses. TXB 2 is thromboxane B 2.
Effect of a6in ~ on the conversion of prosta~landin H 2 by kidney microsomes The preliminary experiments showed that the formation of 6-ketoprostaglandin F . and thromboxane B. is nearly linear during the first 2 min of 2 incubation~'~unde r our assay conditions and this time range was therefore chosen. The effects of aging on the conversion of prostaglandin H to 6-ketoprostaglandin F. and thromboxane B^ are shown in Figs. 3 and 42 A progres• ~ . L . . slve decrease in prostacyclin blosynthesls from prostaglandln H 2 by kidney microsomes was observed. Both of the decreases, from young to mature and from mature to old animals, were statistically significant. However, no significant change in thromboxane biosynthesis from prostaglandin H 2 in kidney microsomes was observed. Effect of asing on the content of fatty acid cyclooxy~enase in kidney The homogeneous sheep seminal vesicular cyclooxygenase was used as standards for the radioimmunoassay of fatty acid cyclooxygenase. The inhibition curve has been reported to be steepest in the range of 5 to 50 ng of standard (3). Therefore, the assays for the samples in the present studies
1274
Aging and Renal Arachidonate Metabolism
120 "
"~
100
o O.
80
Vol. 34, No. 13, 1984
(10)
I
m
E C
60
(10)
II
u_
0
eL
II 40
0
(10)
o I
~0
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t!
20
0
Young
Mature FIG.
Old
3
Effect of age on the biosynthesis of prostacyclin from prostaglandin H_ in kidneys. Values represent the mean + S.E.M. for the number of animals i~dicared in parentheses. 6-Keto PGFI~ is 6-ketoprostaglandin Fld.
Vol. 34, No. 13, 1984
Aging and Renal Arachidonate Metabolism
1275
140(10) 120
(lO) (~o)
100
t
8O
I
mE
CI
E
60
D
C
c~
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i
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20
O"
i
Young
Mature
Old
FIG. 4
Effect of age on the biosynthesis of thromboxane from prostaglandin H^ in kidneys. Values represent the mean + S.E.M. for the number of animals i~dlcared in parentheses. TXB 2 is thromb~xane B 2.
1276
Aging and Renal Arachidonate Metabolism
were performed in this range. acid cyclooxygenase in kidneys observed.
Vol. 34, No. 13, 1984
The effect of aging on the content of fatty is shown in Fig. 5. No significant change was
20
(lO) 18
(lO)
(lO)
÷
16
--t-
14
'°
6 4 2 O
Young
Mature
Old
FIG. 5 Effect of age on the content of fatty acid cyclooxygenase in kidneys. Values represent the mean + S.E.M. for the number of animals indicated in parentheses.
Effect of a~in~ on 15-hydroxyprostaslandin
dehydro~enase
activity in kidney
Fig. 6 shows the effect of aging on 15-hydroxyprostaglandin dehydrogenase activity in kidney cytosol. Kidney cytosol of young rats showed the highest enzyme activity. A progressive decrease as a function of age was found. Both of the decreases in the enzyme activity from young to mature animals, and from mature to old animals, were statistically significant (PCO.OI).
Vol. 34, No. 13, 1984
Aging and Renal Arachidonate Metabolism
1277
6 -
(lO) ~0~
5
X g --
4
ot _ a_ m
3
E E a_ "D
{10)
I
2
4'1 m
) 4u
m
1
<
(10)
o i
Young
Mature
Old
FIG. 6 Effect of age on NAD+-dependent 15-hydroxyprostaglandin dehydrogenase activity in kidneys. Values represent the mean + S.E.M. for the number of animals indicated in parentheses.
Discussion Incubation of cortical and medullary microsomal preparation of rat kidneys with arachidonic acid and prostaglandin H_ shows that both the cortex and medulla could synthesize prostacyclin and t~romboxane (21). Both prostacyclin and thromboxane are also the major products transformed from arachidonic acid by the microsomal preparation of human kidneys, and the biosynthesis of prostacyclin is more prominent than that of prostaglandin E p in human kidneys (7). The thromboxane biosynthesis is of renal parenchymal origin, since the presence of significant thromboxane biosynthesis by cultured glomerular epithelial cells is observed (13). Therefore, the balance between prostacyclin and thromboxane A 2 biosynthesis in kidneys might play an important role in the regulation of renal blood flow, since prostacyclin is a
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Aging and Renal Arachidonate Metabolism
Vol. 34, No. 13, 1984
potent vasodilator and thromboxane Ap a potent vasoconstrictor. In fact, a significant role of renal prostaglanains and thromboxane in the regulation of renal blood flow has been suggested. Blasingham et al. (i) reported that the administration of cyclooxygenase inhlbitors results in decrease in renal blood flow. Morrison et al. (12) have observed that thromboxane A 2 is unmasked in ureterally obstructed perfused kidneys, and thromboxane A 2 might mediate the postobstructive vasoconstriction. In the present investigation, we studied the changes in prostacyclin and thromboxane biosynthetic activities in the microsomal fraction of rat kidneys as a function of age. Conversion of arachidonic acid to prostacyclin progressively decreases with aging (Fig. I), while the formation of thromboxane from arachidonic acid does not significantly change (Fig. 2). In the metabolism of arachidonic acid, it is first converted to prostaglandin endoperoxides (G2, H 2) by fatty acid cyclooxygenase, and then to prostacyclln by prostacyc[in synthetase and to thromboxane A 2 by thromboxane synthetase. If the decrease in prostacyclin biosynthesis in rat kidney with aging is due to a defect in fatty acid cyclooxygenase, it is reasonable to expect that a decrease in thromboxane synthesis should also be observed. In fact, we only observed the decrease in prostacyclin biosynthesis, not in thromboxane biosynthesis. These results indicate that the decrease in the renal prostacyclin biosynthesis in old animals might be due to a defect in prostacyclln synthetase. In order to check this possibility, prostaglandin H 2 was used as substrate. The conversion of prostaglandin H 2 to prostacyclin by kidney microsomes decreases as a function of age (Fig. 3), while the transformation of prostaglandin H. to thromboxane does not show any significant change (Fig. 4). Moreover, t~e immunoreactive fatty acid cyclooxygenase in kidney microsomes does not show any significant change as a function of age (Fig. 5). We therefore concluded that the decrease in prostacyclin biosynthesis in rat kidney with aging is due to a defect in prostacyclin synthetase, not in fatty acid cyclooxygena~e. The present results in kidneys are consistent with our previous results showing an age-related decrease in prostacyclin synthetase activity in cultured rat aortic smooth muscle cells isolated from mature and old rats (2). NAD+-dependent 15-hydroxyprostaglandin dehydrogenase is the first enzyme to metabolize prostaglandins. Kidney is one of the tissues abundant with this enzyme. Prostacyclin is metabolized by renal 15-hydroxyprostaglandin dehydrogenase as efficiently as prostaglandin E[ and F 2 (20). In the metabolism of thromboxane A_ there is no direct evldence showing the thromboxane A_ metabolism by l~-hydroxyprostaglandin dehydrogenase because the standarl thromboxane A^ is not yet available. However, two results to date strongly indicate that thromboxane A ~ is metabolized by 15-hydroxyprostaglandin dehydrogenase. Dawson et al. ('5) analyzed the metabolites of arachidonic acid in guinea pig lung after immunological challenge, and detected more 15-ketothromboxane B_ than thromboxane B_ itself in the arachidonate metabolites. Kung-chao and Tai (8) found that t~romboxane Bp is a very poor substrate for renal 15-hydroxyprostaglandin dehydrogenase. Therefore, it is reasonable to assume that thromboxane A^ is first metabolized by 15-hydroxyprostaglandin dehydrogenase to 15-keto-t~romboxane A 2 and then non-enzymatically converted to 15-keto-thromboxane B^. Therefore, the change in renal 15-hydroxyprostaglandin dehydrogenase activity with aging might have some implications in the physiological function of kidney. The enzyme activity is the highest in young animals (Fig. 6). Since the renal prostacyclin and thromboxane biosynthetic activities are relatively high in young animals, both the prostacyclin
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Aging and Renal Arachidonate Metabolism
1279
and thromboxane A 2 biosynthesized might be equally metabolized by 15-hydroxyprostaglandin deKydrogenase. Consequently, the ratio of renal prostacyclin/ thromboxane A 2 might remain unchanged in young animals. In old animals, the renal prostacyclin biosynthesis is very low, while the renal thromboxane biosynthesis is as active as that in young animals. The decrease in the renal 15-hydroxyprostaglandin dehydrogenase activity might prolong the vasoconstrictive effect of thromboxane A 2. Consequently, it might slow the renal blood flow in old animals. In summary, the present results, indicating the decrease in renal prostacyclin biosynthesis and renal 15-hydroxyprostaglandin dehydrogenase activity with aging, might contribute a plausible explanation of progressive decreases of renal functions in the elderly. Further investigation on the direct involvement of prostacyclin and thromboxane A 2 in the renal functions in vivo in different ages of animals is needed.
Acknowledsements The authors wish to thank the National Institute on Aging, NIH for the supply of animals and to Mrs. Sharon Parker for her excellent technical assistance. Thanks are also due to Mr. Rong-Fong Shen for his assistance in the preparation of prostaglandin H2, and to The Upjohn Co., Kalamazoo, MI, U.S.A. and Ono Pharmaceutical Co., Osaka, Japan for the supply of prostaglandin and thromboxane B 2 standards. We are indebted to MrSw~s. West for her assistance in the preparation of the manuscript. This work supported in part from the American Heart Association Kentucky Affiliate, Inc., the University of Kentucky Isabell L. Kircher Fund, the National Institutes of Health (GM 30380) and the American Heart Association (81-1152).
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i0. II. 12.
M. C. BLASINGHAM, R. E. SHADE, L. SHARE and A. NASJLETTI, J. Pharmacol. Exp. Ther. 214 i-4 (1980). W. C. CHANG, S. MUROTA, J. NAKAO and H. ORIMO, Biochim. Biophys. Acta 620 159-166 (1980). W. C. CHANG, J. NAKAO, S. MUROTA and H. H. TAI, Prostaglandins Leukotrienes Med. I0 33-37 (1983). W. C. CHANG and H. H. TAI, Prostaglandins Leukotrienes Med. (in press). W. DAWSON, J. R. BOOT, A. F. COCKERILL, D. N. B. MALLEN and D. J. OSBORNE, Nature 262 699-702 (1976). M. HAMBERG, J. SVENSSON, T. WAKABAYASHI and B. SAMUELSSON, Proc. Natl. Acad. Sci. USA 71 345-349 (1974). A. HASSID and M. J. DUNN, J. Biol. Chem. 255 2472-2475 (1980). D. T. Y. KUNG-CHAO and H. H. TAI, Biochim. Biophys. Acta 614 1-13 (1980) R. D. LINDEMAN, In The Physiology and Pathology of Human Aging (R. Goldman and M. Rockstein, ed.) pp. 19-38, Academic Press Inc., New York, San Francisco, London (1975). O. H. LOWRY, N. J. ROSENBROUGH, A. L. FARR and R. J. RANDALL, J. Biol. Chem. 193 265-275 (1951). S. MONCADA and J. R. VANE, Pharmacol. Rev. 30 293-331 (1979). A. R. MORRISON, K. NISHIKAWA and P. NEEDLEMAN, J. Pharmacol. Exp. Ther. 205 i-8 (1978).
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A. PETRULIS, M. AIKAWA and M. J. DUNN, Kidney Int. 20 469-474 (1981). G. J. ROTH and E. T. MACHUGA, J. Lab. Clin. Med. 99 187-196 (1982). W. L. SMITH and G. P. WILKIN, Prostaglandins 1 3 873-892 (1977). C. L. TAI and H. H. TAI, Prostaglandins Med. 4 399-408 (1980). H. H. TAI, Biochemistry 15 4586-4592 (1976). H. H. TAI, C. L. TAI and W. L. SMITH, Fed. Proc. 39 323 (1980). H. H. TAI and B. YUAN, Anal. Biochem. 87 343-349 ('[978). P. Y. K. WONG, J. C. McGIFF, L. CAGEN, K. U. MALIK and F. F. SUN, J. Biol. Chem. 254 12-14 (1979). T. V. ZENSER, C. A. HERMAN, R. R. GORMAN and B. B. DAVIS, Biochem. Biophys. Res. Commun. 79 357-363 (1977).