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Metabolism of prostaglandin A1 by hepatic microsomal monooxygenase P-450 system in the guinea pig and rat
Life Sciences Vol . 18, pp . Priated is the U .S .A .
507-514
Pergamon Press
METABOLISM OF PROSTAGLANDIN Al BY HEPATIC MICROSOMAL MONOOXYGENASS P-450 SYSTEM IN THE GUINEA PIG AND RAT David Kupfer and Javier Navarro * The Worcester Foundation for Experimental Biology Shrewsbury, Massachusetts 01545 (Received in final form Jaauary 30, 1976)
Summary This study demonstrates the metabolic transformation of prostaglandin Al (PGA1) by guinea pig and rat liver microsames . The transformation, which required NADPH and oxygen, yielded polar (presumably hydroxylated) products . Incubations with guinea pig liver microsomes yielded one zone of product on tlc, whereas rat liver microsomes produced two discernable metabolic zones . It was demonstrated that PGA1 metabolism in the guinea pig and the rat was inhibited by the addition of SRF-525A, metyrapone, carbon monoxide and cytochrome C ; nicotinamide (10 mM) inhibited only the guinea pig system . These findings indicate that the enzymatic activity responsible for PGA1 metabolism is composed of a typical cytochrome P-450 monooxygenase system . Previous studies demonstrated that guinea pig liver microsomes catalyze the w- and w-1 hydroxylation of prostaglandin A1 (PGAl), yielding 19-hydroxy and 20-hydroxy PGA1 derivatives (1) . Similarly, human liver microso~mes were found to exhibit whydroxylation of PGAl, however no such activity was detected with rat liver preparations (2,3) . On the other hand, studies in vivo in the rat demonstrated that prostaglandins (PGs) are mete lized by w-oxygenation as evidenced by the urinary excretion of w-oxygenated products (2,3) . Questions concerning the nature of the enzyme system involved in the oxidative metabolism of PGs have not yet been answered . Recently we presented suggestive evidence that PGAs are metabolized by the microsomal P-450-monooxygenase system (4) . The evidence obtained was indirect, being based on spectral *This study was carried out in partial fulfillment by J .N . of a Master of Science degree at Cayetano Heredia Peruvian University . Partial support to one of us ES-00834 .
(D .R .) was obtained from NIH Grant
508
PGA1 Metabolism by P-450 Monooaygeaeae
pol . 18, No . 5
binding characteristics of PGs with liver microsomes (4,14) and on observation that PGAl competitively inhibited the in vitro hydroxylation of hexobarbital . The present investigationprovides more direct evidence that the metabolism of PGAl to polar metabolites by liver microsomes, involves catalysis by a cytochrome P-450 monooxygenase system . Furthermore, in contrast to the previously reported inability of rat liver microsomes to metabolize PGAl (2, 3), we observed significant metabolism of PGAl by rat liver microIn addition, our findings showed that the rat liver microsomes . somal system also exhibited a typical monooxygenase activity . Materials and Methods Materials : Prostaglandin Al (5,6- 3 H), 80-100 Ci/mMole was purc asTi~e mom New England Nuclear (Boston, Mass .) . Prostaglandin Al was a gift from Upjohn Co . through the courtesy of Dr . John Pike . Glucose-6-phosphate dehydrogenase E .C . 1 .1 .1 .49, D-Glucose6-phosphate monosodium salt, NADPH monosodium, cytochrome C type III (horse heart) were purchased from Sigma Chemical Co . (St . Louis, Mo .) . SKF-525A .HC1 (ß-diethylaminoethyl diphenylpropylacetate hydrochloride) was a gift from Smith Rline and French Co . Metyrapone (metopyrone) was obtained from Ciba Co . Nicotinamide was purchased from Calbiochem . Thin layer coated plates (Silica Gel G; E . Merck A .G ., Germany) were purchased from Brinckmann Instruments . Animals : Sprague Dawley male rats weighing 90-100 g were obtained r~a~nC'Fiarles River Breeding Laboratories (Wilmington, Mass .) and male guinea pigs weighing 500-600 g were obtained from Elm Hill Farm (Chelmsford, Mass .) . Microsomes : Liver microsomes were prepared as previously descri ) and were stored under layer of 1 .15$ RC1 at -70oC . Under these conditions of storage, activity remained unchanged for several weeks . Protein determinations were carried out by the Lowry procedure (6), using serum albumin as a standard . Incubation : 1 ml of a final volume contained sodium phosphate u fer pH 7 .4, 50 mM), MgC12 (12 .25 mM), microsomel preparation (for protein concentration see text) and prostaglandin Al [5,6-3H] containing additional nonlabeled PGAl (0 .2 mM, ca . 500,000 dpm) . The reaction was usually started by adding the NADPH generating system consisting of glucose-6-phosphate (38 .4 mM), NADP (1 .3 mM) and glucose-6-phosphate dehydrogenase (3 units) or NADPH alone (2 .3 or 3 .4 mM) in few instances . The incubations were carried out by shaking in a Dubnoff incubator at 37oC, in an atmosphere of air . To terminate the reaction and simultaneously to achieve conversion of PGAl and metabolites to the corresponding PGBl derivatives, 0 .5 ml aliquots of aqueous NaOH (4N) were added; the
*EDTA (1mM), which inhibits microsomel lipid peroxidation and prolongs the linearity of monooxygenase activity (7,8,9), had no effect on PGAl metabolism and was left out in subsequent incubations .
Vol. 18, No . 5
PGAl Metabolism by P-450 Moaoorygeaaea
50?
resulting solution was kept at 37°C for an additional 30 minutes* , then was acidified with HC1 to pß.2-3 . The resulting solutions were extracted with 6-volumes of ethyl acetate and the organic phase was removed and evaporated under a stream of N2 with mild heating . The recoveries of the radioactivity were usually 85-958 . The dry samples were dissolved in a few drops of methanol and chra~matographed on thin layer plates (previously activated by heating at 110° for 30 min .) . The solvent system consisted of ethyl acetate-acetic acid-2,2,4-trimethylpentane-water 110 :10 :20 : 100 (10) . The plates were dried in air . The gel in one cm segments was scraped into scintillation vials . One ml methanol was added, the vials were swirled and 5 ml Liquiflor (NEN) was added. The radioactivity was determined in a scintillation spectrometer and the amount of product formed in each zone was determined from the percent of radioactivity in this zone . Results and Discussion Incubation of 3H-PGA1 with guinea pig liver microsomes in the presence of NADPH (~en~erated or added) yielded polar PGA1 metabolite(s) . The chromatographic pattern of the radioactive compounds in the extract from the incubation with guinea pig liver microsomes is demonstrated in Fiq . lA . Since both PGA1 and metabolites have been exposed to ROH, the chromatographic pattern actually reflects mobility of PGB1 derivatives . Two radioactive peaks have been observed : one at 12 cm from the origin reflecting residual parent compound (in the absence of NADPH or microsomes, only this peak appeared ** ) and one at 6 cm from the origin reflecting metabolite (s) (referred to as eak I) . We observed that incubation of PGAl with guinea pig 1 v~icrosomes under conditions reported by Israelsson et al . (1) yielded polar metabolite s) which were chromatograpFiic~ly identical to peak I obtained under our incubation conditions . Since Israelsson et al . (1) demonstrated that the polar PGPil metabolites were 19-hy~roxy and 20-hydroxy PGAl derivatives which did not separate by thin layer chromatography, we assumed that peak I probably contained both metabolites . The characteristics of the guinea pig microsomel enzymatic activity responsible for the transformation of PGA1 was investigated (Tables I and II) . Our results demonstrate that the enzy matic activity is characteristic of a typical cytochrome P-450 mediated monooxygenase . Activity was markedly diminished by inhibitors of monooxygenase such as SRF-525A, metyrapone and nicotinamide (12,13) . As expected, NADPH was required for activity, whereas cytochrome C (0 .5 mM) totally inhibited PGA1 metabolism, suggesting the participation of NADPH-cytochrome C reductase
* In preliminary studies, we determined that conversion of PGAl to PGB , as observed by the appearance of absorbance at 278 nm (10,11 , was complete within 30 min . **When 3H-PGA1 was not incubated but was converted to PGB1 by base treatment, the same chromatographic peak was observed . There was only one detectable peak, demonstrating radiochemical purity of 98+8 . '
PGA1 Mstaboliam by P-450 Mcnooaygeaase
510
vol . ls, xo . 5
10
N
U
i4 U
10
a
s
.
~o
~.
Di~tanos (om)
~
o
â
Di~tanoe(mv
FIG. 1 Chromatographic pattern of t~extract from incubation of PGAl (NADPH generated) with liver microsomes from : uc~ inea ~i ~s (A) and rats (B) . Transformation into PGB1 derivatives and chromatography were performed as described in text . TABLE 1 Effect of Inhibitors of Monooxygenase on PGAl Metabolism by Guinea Pig Liver Microaomes
+ + + -
m nJmg protein d t ons + or nmoles product Deletions (-) (Control) 7 .55 3 .35 SIB-525A 2 .87 Metyrapone Nicotinamide 3 .75 0 Cytochrome Ç 0 NADPH generating system
Inhibition ($) 55 .6 62 .0 50 .3 100 .0 100 .0
Values represent mean of duplicate incubations agreeing within 58 of each other . Conditions are as described in Materials and Methods ; each incubation contained 5 .5 mg of microsomal protein . SIQ'-525A (1 mM) ; Nicotinamide (10 mM) ; Metyrapone (5 mM) and Cytochrome C (0 .5 mM) . When 0 .5 mM concentration of 51~-525A was utilize, inhibition of 25$ was observed .
PGA1 Metabolism bq P-450 Moaoorygeaaea
Vol . 18, No . 5
TABLE 2 Effect of CO on PGA1 Metabolism by Guinea Pig Liver Microsomes nmoles praduat/45 min/mg/protein
$ inhibition
1.
Air
9 .20
-
2.
Air :CO (1 :1) *
1 .70
81 .2
Each incubation contained 6 .3 mg of microsomal protein . *The ratio of CO to air was an approximate value ; care was taken that both incubations received the same amount of air . in this metabolic process . (Table 2) .
Inhibition was also observed with CO
Contrary to previously reported findings that rat .liver microsa~mes do not metabolize PGA1 (2,3), we observed a significant transformation of PGA1 to polar metabolites by liver microsomal preparation from male rats (Fig . 1B) . However, by contrast to the guinea pig, chromatography of the extract from incubations of rat liver microsomes demonstrated two polar metabolic peaks (referred to as I and II) . The more polar peak (I) has an identical Rf as peak I from guinea pig liver incubations . As we found in the guinea pig (Tables l, 2) the formation of peak I in rat liver incubations appears to be mediated by a monooxygenase (Tables 3,4) . This enzymatic activity also requires NADPH and is inhibited by $RF-525A, metyrapone, cytochrome C and carbon monoxide . The only exception is the lack of inhibitory effect by nicotinamide (10 mM) in incubations with rat liver microsomes . The formation of peak II does not appear to be mediated by a monooxygenase as demonstrated by the lack of effect on its formation by the various inhibitors of monooxygenase . TABLE 3 Effect of Inhibitors of Monooxygenase on PGAl Metabolism by Rat Liver Microsomes Metabolites Additions (+j or I II Deletions (-) (nmoles product/90 min/mqprotein) (Control) 6 .40 4 .0 + SRF-525A 2 .74 (57) 3 .7 + Metyrapone 1 .76 (72) 3 .7 + Nicotinamide 7 .55 4 .0 + Cytochrome C 0 (100) -* + NADPH 0 (100) -* Generating System Values represent the mean of duplicate incubations agreeing within 58 of each other; values in parentheses represent 8 inhibition . Microsamal protein was 5 .7 mq per incubation . Additions as in Table 1 . *Was difficult to quantify accurately because of poor definition of the peak .
512
PGA1 Metabolism by P-450 Monoozggenase
Vol . 18, No . 5
TABLE 4 Effect of CO on PGAl Metabolism by Rat Liver Microsomes I
Metabolites
II (nmoles product/90 min/mg protein) Control 02 :N2 (1 :9)
6 .13
3 .90
C0 :02 :N2 (5 :1 :4)
3 .70 (408)
3 .75
Values represent a mean of duplicate incubations ; in parentheses, percent inhibition . Each incubation con twined 5 .85 mg of microsomal protein . The formation of peak I in incubations of PGA1 with guinea pig and rat liver microsomes required oxygen ; little or no metabolism was observed when nitrogen atmosphere was utilized . The previous observations (2,3) that rat liver microsomes do not metabolize PGA1 are difficult to reconcile with our findings . It is possible, however, that the differences in conditions em ployed in the preparation of microsomes and in the incubations in the two respective studies are responsible for the differences in results . In conclusion our studies demonstrate that the transformation of PGA1 into polar metabolites by guinea pig and rat liver microsomes is catalyzed by a typical cytochrome P-450-monooxygenase system . Studies are in progress to further characterize the enzyme system and to identify the metabolites . Acknowledgements The authors greatly appreciate the arrangements made by Dr . F . Welsch which made this study possible ; we acknowledge also the Fullbright Commission's support . The Travel Award to one of us (N .N .) by the Ford Foundation is gratefully acknowledged . References 1. 2. 3. 4. 5. 6. 7. 8. 9.
U . ISRAELSSON, M. FIAMBERG, and B . SAMUELSSON, Europ . J . Biochem . 11, 390-394 (1969) . B . SAMULLSSON, Proc . 4th Internat . Congr . Pharmacol . 12-31 (1970) . B . SAMUELSSON, E . GRANSTRÜM, R. GREEN, and M . BAMBERG, Ann . N .Y . Acad . Sci . 180, 138-163 (1971) . D . KUPFER, Life Sci . 15, 657-670 (1974) . S . BURSTEIN and D . KUPFER, Ann . N .Y . Acad . Sci . _191, 61-67 (1971) . D .H . LOWRY, N .J . ROSEBROUGH, A .L . FARR, and R.J . RANDALL, J . Biol . Chem . 193, 265-275 (1951) . M . JACOBSON, W. LEVIN, A .Y .B . LU, A.H . CONNEY, and R. RUNTZMAN, Drug Metab. Disp . 11, 766-774 (1973) . R .P . VATSIS, J .A . ROWALCHYR, an~M .P . SCBULMAN, Biochem. Biophys . Res . Comet. 61, 258-264 (1974) . E . JEFFREY and G,J, MPiNNERING, Molec, Pharmacol . 10, 10041008 (1974) . '
Vol . 18, No . 5
10 . 11 . 12 . 13 . 14 .
PGA1 Metabolism by P-450 Monoo~geaase
513
M . BYGDEMAN, R. SVANBORG, and B . SAMUELSSON, Clin . Chien . Acte 26, 373-379 (1969) . R.L . JÔNES, J, Lipid Ree . 13, 511-518 (1972) . M .W. ANDERS, Ann . Rev . Pharmacol . ll, 37-56 (1971) . J . SCHENIQIAN, J .A . BALL, and' R.oP . LSTABROOR, Biochem . Pharmacol . 16, 1071-1081 (1967) . J .B . SCHENIQlAN, H . REMMER, and R.W. ~ESTABROOR, Mol . Pharmacol . 3, 113-123 (1967) .
507-514
Pergamon Press
METABOLISM OF PROSTAGLANDIN Al BY HEPATIC MICROSOMAL MONOOXYGENASS P-450 SYSTEM IN THE GUINEA PIG AND RAT David Kupfer and Javier Navarro * The Worcester Foundation for Experimental Biology Shrewsbury, Massachusetts 01545 (Received in final form Jaauary 30, 1976)
Summary This study demonstrates the metabolic transformation of prostaglandin Al (PGA1) by guinea pig and rat liver microsames . The transformation, which required NADPH and oxygen, yielded polar (presumably hydroxylated) products . Incubations with guinea pig liver microsomes yielded one zone of product on tlc, whereas rat liver microsomes produced two discernable metabolic zones . It was demonstrated that PGA1 metabolism in the guinea pig and the rat was inhibited by the addition of SRF-525A, metyrapone, carbon monoxide and cytochrome C ; nicotinamide (10 mM) inhibited only the guinea pig system . These findings indicate that the enzymatic activity responsible for PGA1 metabolism is composed of a typical cytochrome P-450 monooxygenase system . Previous studies demonstrated that guinea pig liver microsomes catalyze the w- and w-1 hydroxylation of prostaglandin A1 (PGAl), yielding 19-hydroxy and 20-hydroxy PGA1 derivatives (1) . Similarly, human liver microso~mes were found to exhibit whydroxylation of PGAl, however no such activity was detected with rat liver preparations (2,3) . On the other hand, studies in vivo in the rat demonstrated that prostaglandins (PGs) are mete lized by w-oxygenation as evidenced by the urinary excretion of w-oxygenated products (2,3) . Questions concerning the nature of the enzyme system involved in the oxidative metabolism of PGs have not yet been answered . Recently we presented suggestive evidence that PGAs are metabolized by the microsomal P-450-monooxygenase system (4) . The evidence obtained was indirect, being based on spectral *This study was carried out in partial fulfillment by J .N . of a Master of Science degree at Cayetano Heredia Peruvian University . Partial support to one of us ES-00834 .
(D .R .) was obtained from NIH Grant
508
PGA1 Metabolism by P-450 Monooaygeaeae
pol . 18, No . 5
binding characteristics of PGs with liver microsomes (4,14) and on observation that PGAl competitively inhibited the in vitro hydroxylation of hexobarbital . The present investigationprovides more direct evidence that the metabolism of PGAl to polar metabolites by liver microsomes, involves catalysis by a cytochrome P-450 monooxygenase system . Furthermore, in contrast to the previously reported inability of rat liver microsomes to metabolize PGAl (2, 3), we observed significant metabolism of PGAl by rat liver microIn addition, our findings showed that the rat liver microsomes . somal system also exhibited a typical monooxygenase activity . Materials and Methods Materials : Prostaglandin Al (5,6- 3 H), 80-100 Ci/mMole was purc asTi~e mom New England Nuclear (Boston, Mass .) . Prostaglandin Al was a gift from Upjohn Co . through the courtesy of Dr . John Pike . Glucose-6-phosphate dehydrogenase E .C . 1 .1 .1 .49, D-Glucose6-phosphate monosodium salt, NADPH monosodium, cytochrome C type III (horse heart) were purchased from Sigma Chemical Co . (St . Louis, Mo .) . SKF-525A .HC1 (ß-diethylaminoethyl diphenylpropylacetate hydrochloride) was a gift from Smith Rline and French Co . Metyrapone (metopyrone) was obtained from Ciba Co . Nicotinamide was purchased from Calbiochem . Thin layer coated plates (Silica Gel G; E . Merck A .G ., Germany) were purchased from Brinckmann Instruments . Animals : Sprague Dawley male rats weighing 90-100 g were obtained r~a~nC'Fiarles River Breeding Laboratories (Wilmington, Mass .) and male guinea pigs weighing 500-600 g were obtained from Elm Hill Farm (Chelmsford, Mass .) . Microsomes : Liver microsomes were prepared as previously descri ) and were stored under layer of 1 .15$ RC1 at -70oC . Under these conditions of storage, activity remained unchanged for several weeks . Protein determinations were carried out by the Lowry procedure (6), using serum albumin as a standard . Incubation : 1 ml of a final volume contained sodium phosphate u fer pH 7 .4, 50 mM), MgC12 (12 .25 mM), microsomel preparation (for protein concentration see text) and prostaglandin Al [5,6-3H] containing additional nonlabeled PGAl (0 .2 mM, ca . 500,000 dpm) . The reaction was usually started by adding the NADPH generating system consisting of glucose-6-phosphate (38 .4 mM), NADP (1 .3 mM) and glucose-6-phosphate dehydrogenase (3 units) or NADPH alone (2 .3 or 3 .4 mM) in few instances . The incubations were carried out by shaking in a Dubnoff incubator at 37oC, in an atmosphere of air . To terminate the reaction and simultaneously to achieve conversion of PGAl and metabolites to the corresponding PGBl derivatives, 0 .5 ml aliquots of aqueous NaOH (4N) were added; the
*EDTA (1mM), which inhibits microsomel lipid peroxidation and prolongs the linearity of monooxygenase activity (7,8,9), had no effect on PGAl metabolism and was left out in subsequent incubations .
Vol. 18, No . 5
PGAl Metabolism by P-450 Moaoorygeaaea
50?
resulting solution was kept at 37°C for an additional 30 minutes* , then was acidified with HC1 to pß.2-3 . The resulting solutions were extracted with 6-volumes of ethyl acetate and the organic phase was removed and evaporated under a stream of N2 with mild heating . The recoveries of the radioactivity were usually 85-958 . The dry samples were dissolved in a few drops of methanol and chra~matographed on thin layer plates (previously activated by heating at 110° for 30 min .) . The solvent system consisted of ethyl acetate-acetic acid-2,2,4-trimethylpentane-water 110 :10 :20 : 100 (10) . The plates were dried in air . The gel in one cm segments was scraped into scintillation vials . One ml methanol was added, the vials were swirled and 5 ml Liquiflor (NEN) was added. The radioactivity was determined in a scintillation spectrometer and the amount of product formed in each zone was determined from the percent of radioactivity in this zone . Results and Discussion Incubation of 3H-PGA1 with guinea pig liver microsomes in the presence of NADPH (~en~erated or added) yielded polar PGA1 metabolite(s) . The chromatographic pattern of the radioactive compounds in the extract from the incubation with guinea pig liver microsomes is demonstrated in Fiq . lA . Since both PGA1 and metabolites have been exposed to ROH, the chromatographic pattern actually reflects mobility of PGB1 derivatives . Two radioactive peaks have been observed : one at 12 cm from the origin reflecting residual parent compound (in the absence of NADPH or microsomes, only this peak appeared ** ) and one at 6 cm from the origin reflecting metabolite (s) (referred to as eak I) . We observed that incubation of PGAl with guinea pig 1 v~icrosomes under conditions reported by Israelsson et al . (1) yielded polar metabolite s) which were chromatograpFiic~ly identical to peak I obtained under our incubation conditions . Since Israelsson et al . (1) demonstrated that the polar PGPil metabolites were 19-hy~roxy and 20-hydroxy PGAl derivatives which did not separate by thin layer chromatography, we assumed that peak I probably contained both metabolites . The characteristics of the guinea pig microsomel enzymatic activity responsible for the transformation of PGA1 was investigated (Tables I and II) . Our results demonstrate that the enzy matic activity is characteristic of a typical cytochrome P-450 mediated monooxygenase . Activity was markedly diminished by inhibitors of monooxygenase such as SRF-525A, metyrapone and nicotinamide (12,13) . As expected, NADPH was required for activity, whereas cytochrome C (0 .5 mM) totally inhibited PGA1 metabolism, suggesting the participation of NADPH-cytochrome C reductase
* In preliminary studies, we determined that conversion of PGAl to PGB , as observed by the appearance of absorbance at 278 nm (10,11 , was complete within 30 min . **When 3H-PGA1 was not incubated but was converted to PGB1 by base treatment, the same chromatographic peak was observed . There was only one detectable peak, demonstrating radiochemical purity of 98+8 . '
PGA1 Mstaboliam by P-450 Mcnooaygeaase
510
vol . ls, xo . 5
10
N
U
i4 U
10
a
s
.
~o
~.
Di~tanos (om)
~
o
â
Di~tanoe(mv
FIG. 1 Chromatographic pattern of t~extract from incubation of PGAl (NADPH generated) with liver microsomes from : uc~ inea ~i ~s (A) and rats (B) . Transformation into PGB1 derivatives and chromatography were performed as described in text . TABLE 1 Effect of Inhibitors of Monooxygenase on PGAl Metabolism by Guinea Pig Liver Microaomes
+ + + -
m nJmg protein d t ons + or nmoles product Deletions (-) (Control) 7 .55 3 .35 SIB-525A 2 .87 Metyrapone Nicotinamide 3 .75 0 Cytochrome Ç 0 NADPH generating system
Inhibition ($) 55 .6 62 .0 50 .3 100 .0 100 .0
Values represent mean of duplicate incubations agreeing within 58 of each other . Conditions are as described in Materials and Methods ; each incubation contained 5 .5 mg of microsomal protein . SIQ'-525A (1 mM) ; Nicotinamide (10 mM) ; Metyrapone (5 mM) and Cytochrome C (0 .5 mM) . When 0 .5 mM concentration of 51~-525A was utilize, inhibition of 25$ was observed .
PGA1 Metabolism bq P-450 Moaoorygeaaea
Vol . 18, No . 5
TABLE 2 Effect of CO on PGA1 Metabolism by Guinea Pig Liver Microsomes nmoles praduat/45 min/mg/protein
$ inhibition
1.
Air
9 .20
-
2.
Air :CO (1 :1) *
1 .70
81 .2
Each incubation contained 6 .3 mg of microsomal protein . *The ratio of CO to air was an approximate value ; care was taken that both incubations received the same amount of air . in this metabolic process . (Table 2) .
Inhibition was also observed with CO
Contrary to previously reported findings that rat .liver microsa~mes do not metabolize PGA1 (2,3), we observed a significant transformation of PGA1 to polar metabolites by liver microsomal preparation from male rats (Fig . 1B) . However, by contrast to the guinea pig, chromatography of the extract from incubations of rat liver microsomes demonstrated two polar metabolic peaks (referred to as I and II) . The more polar peak (I) has an identical Rf as peak I from guinea pig liver incubations . As we found in the guinea pig (Tables l, 2) the formation of peak I in rat liver incubations appears to be mediated by a monooxygenase (Tables 3,4) . This enzymatic activity also requires NADPH and is inhibited by $RF-525A, metyrapone, cytochrome C and carbon monoxide . The only exception is the lack of inhibitory effect by nicotinamide (10 mM) in incubations with rat liver microsomes . The formation of peak II does not appear to be mediated by a monooxygenase as demonstrated by the lack of effect on its formation by the various inhibitors of monooxygenase . TABLE 3 Effect of Inhibitors of Monooxygenase on PGAl Metabolism by Rat Liver Microsomes Metabolites Additions (+j or I II Deletions (-) (nmoles product/90 min/mqprotein) (Control) 6 .40 4 .0 + SRF-525A 2 .74 (57) 3 .7 + Metyrapone 1 .76 (72) 3 .7 + Nicotinamide 7 .55 4 .0 + Cytochrome C 0 (100) -* + NADPH 0 (100) -* Generating System Values represent the mean of duplicate incubations agreeing within 58 of each other; values in parentheses represent 8 inhibition . Microsamal protein was 5 .7 mq per incubation . Additions as in Table 1 . *Was difficult to quantify accurately because of poor definition of the peak .
512
PGA1 Metabolism by P-450 Monoozggenase
Vol . 18, No . 5
TABLE 4 Effect of CO on PGAl Metabolism by Rat Liver Microsomes I
Metabolites
II (nmoles product/90 min/mg protein) Control 02 :N2 (1 :9)
6 .13
3 .90
C0 :02 :N2 (5 :1 :4)
3 .70 (408)
3 .75
Values represent a mean of duplicate incubations ; in parentheses, percent inhibition . Each incubation con twined 5 .85 mg of microsomal protein . The formation of peak I in incubations of PGA1 with guinea pig and rat liver microsomes required oxygen ; little or no metabolism was observed when nitrogen atmosphere was utilized . The previous observations (2,3) that rat liver microsomes do not metabolize PGA1 are difficult to reconcile with our findings . It is possible, however, that the differences in conditions em ployed in the preparation of microsomes and in the incubations in the two respective studies are responsible for the differences in results . In conclusion our studies demonstrate that the transformation of PGA1 into polar metabolites by guinea pig and rat liver microsomes is catalyzed by a typical cytochrome P-450-monooxygenase system . Studies are in progress to further characterize the enzyme system and to identify the metabolites . Acknowledgements The authors greatly appreciate the arrangements made by Dr . F . Welsch which made this study possible ; we acknowledge also the Fullbright Commission's support . The Travel Award to one of us (N .N .) by the Ford Foundation is gratefully acknowledged . References 1. 2. 3. 4. 5. 6. 7. 8. 9.
U . ISRAELSSON, M. FIAMBERG, and B . SAMUELSSON, Europ . J . Biochem . 11, 390-394 (1969) . B . SAMULLSSON, Proc . 4th Internat . Congr . Pharmacol . 12-31 (1970) . B . SAMUELSSON, E . GRANSTRÜM, R. GREEN, and M . BAMBERG, Ann . N .Y . Acad . Sci . 180, 138-163 (1971) . D . KUPFER, Life Sci . 15, 657-670 (1974) . S . BURSTEIN and D . KUPFER, Ann . N .Y . Acad . Sci . _191, 61-67 (1971) . D .H . LOWRY, N .J . ROSEBROUGH, A .L . FARR, and R.J . RANDALL, J . Biol . Chem . 193, 265-275 (1951) . M . JACOBSON, W. LEVIN, A .Y .B . LU, A.H . CONNEY, and R. RUNTZMAN, Drug Metab. Disp . 11, 766-774 (1973) . R .P . VATSIS, J .A . ROWALCHYR, an~M .P . SCBULMAN, Biochem. Biophys . Res . Comet. 61, 258-264 (1974) . E . JEFFREY and G,J, MPiNNERING, Molec, Pharmacol . 10, 10041008 (1974) . '
Vol . 18, No . 5
10 . 11 . 12 . 13 . 14 .
PGA1 Metabolism by P-450 Monoo~geaase
513
M . BYGDEMAN, R. SVANBORG, and B . SAMUELSSON, Clin . Chien . Acte 26, 373-379 (1969) . R.L . JÔNES, J, Lipid Ree . 13, 511-518 (1972) . M .W. ANDERS, Ann . Rev . Pharmacol . ll, 37-56 (1971) . J . SCHENIQIAN, J .A . BALL, and' R.oP . LSTABROOR, Biochem . Pharmacol . 16, 1071-1081 (1967) . J .B . SCHENIQlAN, H . REMMER, and R.W. ~ESTABROOR, Mol . Pharmacol . 3, 113-123 (1967) .