Antioxidant Inhibition of Prostaglandin Production by Rat Renal Medulla Terry V. Zenser and Bernard Antioxidant effects upon renal production of both prostaglandins and CAMP were investigated using slices of rat inner medulla. Synthetic antioxidants were more potent inhibitors of prostaglandin production than were naturally occurring ontioxidants. Synthetic compounds 2,7-naphthalenediol, and Santoquin@ (Ethoxyquin) caused a 60% inhibition of prostaglandin Es(PGE,) synthesis at a concentration of 0.01 mM. Ascorbic acid caused only a 30% inhibition at a concentmtion of 10 mM. Antioxidant inhibition of prostoglandin production was also observed following arachidonic acid addition. Antioxidants that reduced PGE, synthesis also reduced PGFs, synthesis. Test agents found to reduce prostaglandin synthesis also lowered CAMP content. 2,7_Naphthalenediol elicited a dose-dependent decrease in
9. Davis
both prostaglandin synthesis and CAMP content. While PGEr did not increase CAMP in control slices, the low CAMP level produced by Santoquin was increased to control values by PGEt. Furthermore, Santoquin and 2,7-naphthalenediol did not alter arginine vasopressin-stimulated CAMP content. By contrast, inhibition of the arginine vasopressin stimulation by butylated hydroxyanisole suggested additional effects by this agent. These results care consistent with the hypothesis that endogenously produced PGb can exert a hormonelike action in the inner medulla by increasing CAMP content. Advantages of the inner medullary slice system compared to homogenates for investigation of the actions of antioxidants or other agents thought to alter prostaglandin synthesis are discussed.
P
ROSTAGLANDINS are biologically active compounds that have been demonstrated to exert physiologic or biochemical effects in a number of tissues, including the kidney. They have been shown to increase the rate of urine flow and sodium excretion. ‘J Prostaglandins also activate renal adenylate cyclase and increase tissue CAMP content. 3,4Within the kidney, prostaglandin synthesis, a process requiring oxygen, proceeds primarily in the inner medulla via the enzyme cyclooxygenase. x This localization of prostaglandin synthesis is of interest because the inner medulla functions in a relatively anaerobic environment,*-” and because oxygen has been shown to increase prostaglandin production. 5-” Changes in inner medullary slice prostaglandin synthesis have been correlated with tissue CAMP content demonstrating a system of local prostaglandin synthesis and action.“*‘2 Substances such as antioxidants that inhibit oxidative reactions might be expected to affect prostaglandin synthesis. However, that possibility has not been extensively examined in preparations utilizing intact cells. Antioxidants are used as food additives because they act as preservatives preventing oxidation of nutrients. They have also been found to delay the aging process in
From the Geriatric Center, Veterans Administration Hospital, and the Departments of Medicine and Biochemistry, St. Louis Universiry. St. Louis, MO. Receivedfor publication April 18, 1977. Supported by the Veterans Adminisrration. Reprint requests should be addressed to Terry V. Zenser. Ph.D., Geriatric Center, VA Hospiral, St. Louis, MO. 63125. @ 1978 by Grune & Stratton. Inc. 0026-0495/78/2702-0011$01.00/0 Metabolism, Vol. 27, No. 2 (February), 1978
227
228
ZENSER
AND DAVIS
animals and are commercially used for this purpose.‘3 I5 In view of the known ability of the kidney to concentrate many substances and its prostaglandin synthetic capacity, the present study was designed to investigate the effect of natural and synthetic antioxidants on prostaglandin synthesis by renal inner medullary slices. Changes in prostaglandin synthesis were correlated with changes in tissue CAMP content. MATERIALS
AND METHODS
‘H-prostaglandin Ez (PGEz; II7 Ci/mmole) and ‘H-PGFla (178 Ci/mmole) were purchased from New England Nuclear, Boston, Mass. Rabbit antisera to either PGE or PGF were obtained from Regis Chemical, Morton Grove, III. Antibodies, Davis, Calif.. provided goat antisera to rabbit gamma globulins. Unlabeled PGE2 and PGF2, were a generous gift of Dr. John Pike, (Santoquin) was Upjohn, Kalamazoo, Mich. 6-Eth oxy-l,2-dihydro-2,2,4-trimethylquinoline kindly donated by Dr. G. L. Romoser, Monsanto. St. Louis. MO. CAMP-binding protein was were purchased in the highest available prepared as described by Gilman.16 All other chemicals grade from standard sources.
Tissue Incubation Sprague-Dawley rats weighing 250.-300 g were anesthetized with diethyl ether. Their kidneys were removed quickly and placed in cold 0.85”,, NaCI. Inner medullary slices were prepared with a Stadie-Riggs microtome as previously described.s Slices were incubated in 2 ml of Krebs-Ringer bicarbonate buffer containing I mg/ml each of glucose and bovine serum albumin. Slices were first incubated for 20 min in a gas phase of 5”,, CO?. 95”,, N2 and then transferred to flasks containing 5% CO?, 95% 02 for the remaining I5 min. Test agents were present during both incubations. Prior to incubation. each flask was purged with gas and the oxygen concentration was verified with a Yellow Springs Instrument model 53 oxygen monitor. Incubations were performed at 37°C with 100 cycle/min agitation in a Dubnoff metabolic incubator. Tissue was removed for CAMP determination, while the incubation medium was immediately frozen at -35°C for later analysis of PGE2 and PGFlu content.
Prostaglandin Analysis Incubation media were generally analyzed within I wk, although it was found that storage at -35°C for more than I mo did not alter the prostaglandin content. A trace amount of tritiated PGF2, or PGE2 (approximately 1000 cpm or 3 pg) was added to each 0.5-ml aliquot of thawed medium. After adjustment to pH 3.0~3.5 with I.0 N HCI, each aliquot was extracted twice with I.5 ml of ethyl acetate. Combined extracts were evaporated to dryness at 40°C under Nz. Dried extracts were dissolved in 0.5 ml of assay buffer consisting of 0.01 M NaHzPOd. 0.15 M NaCI. and I”,, normal rabbit serum, pH 7.0. An aliquot of 0.050 ml was removed to determine the recovery of prostaglandin during extraction (85”,, --95”,,). Reported values were corrected for recovery. Additional aliquots were analyzed in duplicate to determine the prostaglandin content. Radioimmunoassays for PGE2 and PGFza were performed as described previously.s Antistandards or unknown body, plus ‘H-prostaglandin (2000 cpm, 6.0 pg) and prostaglandin (0.05 ml) were incubated in 1.0 ml of pH 7.0 buffer at 4°C. After 4 hr. goat antiserum to rabbit gamma globulins was added and incubation was continued for 20 hr at 4°C. The precipitate was collected by centrifugation at 3000 rpm for 30 min, dissolved in I.0 ml of I.0 N NaOH, and counted. Standards and unknowns were routinely run in duplicate with the reproducibility between duplicate samples averaging 3.4”,,. Unknowns exhibited linearity with respect to sample dilutions and combinations of unknowns and standards were additive. Cross-reactivity of PGE antibody with PGFza or PGD2 or PGF antibody with PGE2 and PGD2 was approximately I”,, at the 50”,, displacement level. Thus, the assay technique could di;;inguish among the three major classes of prostaglandins known to be produced by the kidney. The PGE antibody could not, distinguish PGF,, from however, distinguish PGE, from PGEt , nor could the PGF antibody PGF2,. However, it has been previously reported that only two series of prostaglandins are Experiments utilizing radiolabeled PGEz indicate no detectable produced by the kidney.‘s”9
ANTIOXIDANT
INHIBITION
OF PG PRODUCTION
229
PGEz metabolism by the inner medulla, a finding consistent with previous reports.20 Therefore, it was assumed that under the conditions of these experiments the PGE and PGF antibodies were Logit transformations of the standard curves remeasuring PGE2 and PGFza, respectively. sulted in linear functions between 0.1 and 10 ng prostaglandin/tube. equivalent to 2.0-200 ng prostaglandin/ml of incubation medium. Prostaglandin content was expressed as ng/mg wet weight of tissue.
CAMP Analysis CAMP was extracted by homogenizing slices of 0.5 ml of 50 mM sodium acetate buffer (pH 4.0) at 95°C.” The homogenate was heated for 5 min at that temperature. Cooled homogenates were spun at 3000 rpm for IO min at 4°C. CAMP was determined in the supernatant by addition of 2000 by the protein-binding method of Gilman.16 CAMP recovery was monitored cpm of ‘H-cAMP to the extraction buffer. Each reported result was corrected for recovery and represents the mean f SE of three incubation samples, each analyzed in duplicate. Statistical differences were evaluated by Student’s t test for unpaired values. RESULTS
Effects of the naturally occurring antioxidants tested on prostaglandin production are tabulated in Table 1. While 10 and 20 mM vitamin C were inhibitory, lower concentrations of vitamins C, A, and E were not. Ethanol, which was used as a diluent for vitamin A and E, also did not alter PGEz production. Inhibition was observed with 10 and 20 mM glutathione, but lower concentrations were not inhibitory. Synthetic antioxidants also exhibited a concentration-dependent inhibition of PGEz production (Table 2). Butylated hydroxyanisole inhibited PGEz production at I mM but not at 0.1 or 0.01 mM. Butylated hydroxytoluene (1 mM) did not alter PGEz synthesis. Both 2,7_naphthalenediol and Santoquin elicited a 500: reduction in PGEz production between 0.01 and 0.001 mM. Values for PGEz production reported for 0.1 mM Santoquin and 2,7_naphthalenediol, 1.3 =t 0.6 and 0.8 f 0.1 ng/mg wet weight, respectively, were at the lower limit of our detection. Arachidonic acid is a substrate for prostaglandin synthesis and increases prostaglandin synthesis in several in vitro and in vivo systems. The twofold increase in PGEZ synthesis elicited by 170 &I arachidonic acid was
Table 1. Effects of Naturally Medullarv
Occurring AnGoxidantr Prostaslandin
Concentration (mM 1
on Rat Renal Inner
Production
PGE2 (ng/mg wet weight)
PGFz, (ng/mg wet weight)
Control
14.0 + 1.2
4.5 * 0.4
Ethanol (1.2%. v/v)
14.8 f 0.8
4.3 f 0.3
Vitamin A
1
13.6 + 1.9
Vitamin C
1
12.3 f
10 20 Vitamin E Glutathione
*p valueat least
1.8
9.1 f 2.0* 3.0 f 0.1*
1.5 f 0.2*
1
11.2 i
5
18.1 f 2.2
1
17.2 f 2.0
3.9 f 0.4
10
10.7 f
1.0*
3.1 f 0.1*
20
4.4 i
1.0*
1.3 f 0.2*
9. Davis
both prostaglandin synthesis and CAMP content. While PGEr did not increase CAMP in control slices, the low CAMP level produced by Santoquin was increased to control values by PGEt. Furthermore, Santoquin and 2,7-naphthalenediol did not alter arginine vasopressin-stimulated CAMP content. By contrast, inhibition of the arginine vasopressin stimulation by butylated hydroxyanisole suggested additional effects by this agent. These results care consistent with the hypothesis that endogenously produced PGb can exert a hormonelike action in the inner medulla by increasing CAMP content. Advantages of the inner medullary slice system compared to homogenates for investigation of the actions of antioxidants or other agents thought to alter prostaglandin synthesis are discussed.
P
ROSTAGLANDINS are biologically active compounds that have been demonstrated to exert physiologic or biochemical effects in a number of tissues, including the kidney. They have been shown to increase the rate of urine flow and sodium excretion. ‘J Prostaglandins also activate renal adenylate cyclase and increase tissue CAMP content. 3,4Within the kidney, prostaglandin synthesis, a process requiring oxygen, proceeds primarily in the inner medulla via the enzyme cyclooxygenase. x This localization of prostaglandin synthesis is of interest because the inner medulla functions in a relatively anaerobic environment,*-” and because oxygen has been shown to increase prostaglandin production. 5-” Changes in inner medullary slice prostaglandin synthesis have been correlated with tissue CAMP content demonstrating a system of local prostaglandin synthesis and action.“*‘2 Substances such as antioxidants that inhibit oxidative reactions might be expected to affect prostaglandin synthesis. However, that possibility has not been extensively examined in preparations utilizing intact cells. Antioxidants are used as food additives because they act as preservatives preventing oxidation of nutrients. They have also been found to delay the aging process in
From the Geriatric Center, Veterans Administration Hospital, and the Departments of Medicine and Biochemistry, St. Louis Universiry. St. Louis, MO. Receivedfor publication April 18, 1977. Supported by the Veterans Adminisrration. Reprint requests should be addressed to Terry V. Zenser. Ph.D., Geriatric Center, VA Hospiral, St. Louis, MO. 63125. @ 1978 by Grune & Stratton. Inc. 0026-0495/78/2702-0011$01.00/0 Metabolism, Vol. 27, No. 2 (February), 1978
227
228
ZENSER
AND DAVIS
animals and are commercially used for this purpose.‘3 I5 In view of the known ability of the kidney to concentrate many substances and its prostaglandin synthetic capacity, the present study was designed to investigate the effect of natural and synthetic antioxidants on prostaglandin synthesis by renal inner medullary slices. Changes in prostaglandin synthesis were correlated with changes in tissue CAMP content. MATERIALS
AND METHODS
‘H-prostaglandin Ez (PGEz; II7 Ci/mmole) and ‘H-PGFla (178 Ci/mmole) were purchased from New England Nuclear, Boston, Mass. Rabbit antisera to either PGE or PGF were obtained from Regis Chemical, Morton Grove, III. Antibodies, Davis, Calif.. provided goat antisera to rabbit gamma globulins. Unlabeled PGE2 and PGF2, were a generous gift of Dr. John Pike, (Santoquin) was Upjohn, Kalamazoo, Mich. 6-Eth oxy-l,2-dihydro-2,2,4-trimethylquinoline kindly donated by Dr. G. L. Romoser, Monsanto. St. Louis. MO. CAMP-binding protein was were purchased in the highest available prepared as described by Gilman.16 All other chemicals grade from standard sources.
Tissue Incubation Sprague-Dawley rats weighing 250.-300 g were anesthetized with diethyl ether. Their kidneys were removed quickly and placed in cold 0.85”,, NaCI. Inner medullary slices were prepared with a Stadie-Riggs microtome as previously described.s Slices were incubated in 2 ml of Krebs-Ringer bicarbonate buffer containing I mg/ml each of glucose and bovine serum albumin. Slices were first incubated for 20 min in a gas phase of 5”,, CO?. 95”,, N2 and then transferred to flasks containing 5% CO?, 95% 02 for the remaining I5 min. Test agents were present during both incubations. Prior to incubation. each flask was purged with gas and the oxygen concentration was verified with a Yellow Springs Instrument model 53 oxygen monitor. Incubations were performed at 37°C with 100 cycle/min agitation in a Dubnoff metabolic incubator. Tissue was removed for CAMP determination, while the incubation medium was immediately frozen at -35°C for later analysis of PGE2 and PGFlu content.
Prostaglandin Analysis Incubation media were generally analyzed within I wk, although it was found that storage at -35°C for more than I mo did not alter the prostaglandin content. A trace amount of tritiated PGF2, or PGE2 (approximately 1000 cpm or 3 pg) was added to each 0.5-ml aliquot of thawed medium. After adjustment to pH 3.0~3.5 with I.0 N HCI, each aliquot was extracted twice with I.5 ml of ethyl acetate. Combined extracts were evaporated to dryness at 40°C under Nz. Dried extracts were dissolved in 0.5 ml of assay buffer consisting of 0.01 M NaHzPOd. 0.15 M NaCI. and I”,, normal rabbit serum, pH 7.0. An aliquot of 0.050 ml was removed to determine the recovery of prostaglandin during extraction (85”,, --95”,,). Reported values were corrected for recovery. Additional aliquots were analyzed in duplicate to determine the prostaglandin content. Radioimmunoassays for PGE2 and PGFza were performed as described previously.s Antistandards or unknown body, plus ‘H-prostaglandin (2000 cpm, 6.0 pg) and prostaglandin (0.05 ml) were incubated in 1.0 ml of pH 7.0 buffer at 4°C. After 4 hr. goat antiserum to rabbit gamma globulins was added and incubation was continued for 20 hr at 4°C. The precipitate was collected by centrifugation at 3000 rpm for 30 min, dissolved in I.0 ml of I.0 N NaOH, and counted. Standards and unknowns were routinely run in duplicate with the reproducibility between duplicate samples averaging 3.4”,,. Unknowns exhibited linearity with respect to sample dilutions and combinations of unknowns and standards were additive. Cross-reactivity of PGE antibody with PGFza or PGD2 or PGF antibody with PGE2 and PGD2 was approximately I”,, at the 50”,, displacement level. Thus, the assay technique could di;;inguish among the three major classes of prostaglandins known to be produced by the kidney. The PGE antibody could not, distinguish PGF,, from however, distinguish PGE, from PGEt , nor could the PGF antibody PGF2,. However, it has been previously reported that only two series of prostaglandins are Experiments utilizing radiolabeled PGEz indicate no detectable produced by the kidney.‘s”9
ANTIOXIDANT
INHIBITION
OF PG PRODUCTION
229
PGEz metabolism by the inner medulla, a finding consistent with previous reports.20 Therefore, it was assumed that under the conditions of these experiments the PGE and PGF antibodies were Logit transformations of the standard curves remeasuring PGE2 and PGFza, respectively. sulted in linear functions between 0.1 and 10 ng prostaglandin/tube. equivalent to 2.0-200 ng prostaglandin/ml of incubation medium. Prostaglandin content was expressed as ng/mg wet weight of tissue.
CAMP Analysis CAMP was extracted by homogenizing slices of 0.5 ml of 50 mM sodium acetate buffer (pH 4.0) at 95°C.” The homogenate was heated for 5 min at that temperature. Cooled homogenates were spun at 3000 rpm for IO min at 4°C. CAMP was determined in the supernatant by addition of 2000 by the protein-binding method of Gilman.16 CAMP recovery was monitored cpm of ‘H-cAMP to the extraction buffer. Each reported result was corrected for recovery and represents the mean f SE of three incubation samples, each analyzed in duplicate. Statistical differences were evaluated by Student’s t test for unpaired values. RESULTS
Effects of the naturally occurring antioxidants tested on prostaglandin production are tabulated in Table 1. While 10 and 20 mM vitamin C were inhibitory, lower concentrations of vitamins C, A, and E were not. Ethanol, which was used as a diluent for vitamin A and E, also did not alter PGEz production. Inhibition was observed with 10 and 20 mM glutathione, but lower concentrations were not inhibitory. Synthetic antioxidants also exhibited a concentration-dependent inhibition of PGEz production (Table 2). Butylated hydroxyanisole inhibited PGEz production at I mM but not at 0.1 or 0.01 mM. Butylated hydroxytoluene (1 mM) did not alter PGEz synthesis. Both 2,7_naphthalenediol and Santoquin elicited a 500: reduction in PGEz production between 0.01 and 0.001 mM. Values for PGEz production reported for 0.1 mM Santoquin and 2,7_naphthalenediol, 1.3 =t 0.6 and 0.8 f 0.1 ng/mg wet weight, respectively, were at the lower limit of our detection. Arachidonic acid is a substrate for prostaglandin synthesis and increases prostaglandin synthesis in several in vitro and in vivo systems. The twofold increase in PGEZ synthesis elicited by 170 &I arachidonic acid was
Table 1. Effects of Naturally Medullarv
Occurring AnGoxidantr Prostaslandin
Concentration (mM 1
on Rat Renal Inner
Production
PGE2 (ng/mg wet weight)
PGFz, (ng/mg wet weight)
Control
14.0 + 1.2
4.5 * 0.4
Ethanol (1.2%. v/v)
14.8 f 0.8
4.3 f 0.3
Vitamin A
1
13.6 + 1.9
Vitamin C
1
12.3 f
10 20 Vitamin E Glutathione
*p valueat least
1.8
9.1 f 2.0* 3.0 f 0.1*
1.5 f 0.2*
1
11.2 i
5
18.1 f 2.2
1
17.2 f 2.0
3.9 f 0.4
10
10.7 f
1.0*
3.1 f 0.1*
20
4.4 i
1.0*
1.3 f 0.2*
