PROSTAGLANDINS
STUDIES OF THE MECHANISMS INVOLVED IN THE FATE OF PROSTACYCLIN (PGQ)
AND 6-KETO-PGF,a IN THE
PULMONARY CIRCULATION Hollis J. Hawkins,' J. Bryan Smith* Kyriacos C. Nicolaou,' and Thomas E. Eling' 1 Prostaglandins Section Laboratory of Pulmonary Function and Toxicology National Institute of Environmental Health Sciences Research Triangle Park, North Carolina 27709 *Cardeza Foundation Thomas Jefferson University Philadelphia, Pennsylvania 19107 'Department of Chemistry University of Pennsylvania Philadelphia, Pennsylvania 19104
ABSTRACT We have investigated the metabolism of [3]H_prostaglandin(PG)I2 and its non-enzymatic breakdown product [3]H-6-keto-PGFl,by rat pulmonary tissue and their possible uptake and metabolism upon passage through the isolated perfused rat lung. When incubated with rat lung homogenate in the presence of B-NAD, [3]H-PGI was extensively degraded into at least one metabolite, while [35H-6-keto-PGFl was only minimally metabolized. However, on passage through isolate% perfused rat lungs, neither [3]H-PG12 nor [3]H-6-keto-PGFl were removed from the circulation into the lung or degraded. Tiis demonstration that PG12 is not a substrate for the transport system for the removal of PGs from the circulation into the lung further illustrates that this system is a critical determinant for the pulmonary inactivation of circulating prostaglandins. The experimental findings are discussed in reference to the structure-activityrequirements necessary for pulmonary transport and subsequent metabolism.
DECEMBER 1978 VOL. 16 NO. 6
871
PROSTAGLANDINS
INTRODUCTION Prostacyclin, or prostaglandin (PG)Iz, has recently been shown to be a novel PG biosynthesized from the PG endoperoxides PGG2 and PGH2 (l-4). Studies involving the direct measurement of PG12 or the detection of its single non-enzymatic, biologically inactive breakdown product, 6-keto-PGFl, (5), have shown that PG12 is formed by arteries of various species, human veins, rat stomach fundus, and animal lungs, seminal vesicles and heart (l-4, 6-11). PG12 is the most potent of the PG inhibitors of platelet aggregation yet identified (l-3), and has been shown to be a powerful coronary vasodilator (12-14). The balance between levels of this bicyclic PG and thromboxane (TX) A2 appears to play a critical role in the control of thrombus formation (15). Since the half-life of PG12 in blood at physiological pH (16) is long enough to make it an important circulating hormone, its possible metabolism during passage through the circulation could affect the PGI2-TXA balance. It is well established that the fung plays a major role in the metabolism of circulating PGs (for review see ref. 17). We have previously demonstrated that a transport system is necessary for the passage of PGs into the pulmonary cells for subsequent inactivation by intracellular enzymes, and that the substrate specificity for the inactivation of circulating PGs appears to reside with the transport system (18). An apparent lack of biological inactivation of PG12 on passage through the lung has been suggested by several investigators (13,16, 19). In this paper we have directly studied the pulmonary metabolism of PG12 and 6-keto-PGFl and have investigated the underlying mechanisms responsible for &e lack of inactivation by (i) examining the metabolism of [3]H-PG12 and its hydrolysis product [SJH-6-keto-PGFl, by rat lung homogenates, and (ii) studying the passage of [3]H-PG12 and [3]H-6-keto-PGFl,through the isolated perfused rat lung. MATERIALS AND METHODS [9-(3)H]-PG12 (10.9 Ci/mmole or 12.0 Ci/mmole) was prepared according to the method of Nicolaou -_ et al. (20). [9-(3)-HI-PGF2, (9.2 Ci/mmole) and [14]C - dextran (M.W. - 70,000, 1.043 mCi/gram) were purchased from New England Nuclear, Boston, MA. Authentic standards of PGE2, PGF2,-THAM, 13,14-dihydro-PGFp,,13,14-dihydro15-keto-PGFp,, 15-keto-PGF2,, and 6-keto-PGFl, were gifts from Dr. John Pike, Upjohn Co., Kalamazoo, MI. Dr. Frank Sun of Upjohn CO. kindly provided authentic standards of 6-keto-13,14-dihydro-PGFl., 6,15-diketo-13,14-dihydro-PGFl. and 6,15-diketo-PGFl,. Silica gel G thin layer plates (250~) were purchased from Analtech Inc., Newark, DE. B-NAD was obtained from Sigma Chemical Co., St. Louis, MO. Blue dextran (M.W. -.2 X 106) was purchased from Pharmacia Fine Chemicals, Piscataway, NJ. Lung homogenate incubation mixtures and isolated perfused lung
872
DECEMBER 1978 VOL. 16 NO. 6
PROSTAGLANDINS
(IPL) effluents were analyzed for metabolites by thin layer chromatography (TLC). Samples were acidified to pH 3.0-3.5 with HCl and extracted twice with 6 vol ethyl acetate or ether, and the extracts were evaporated to dryness in vacua. The residues were dissolved in methanol or acetone and ali?$ioEand unlabelled standards were applied to silica gel plates which were developed in chloroform, methanol, acetic acid, water (135:12:1.5:1.2)(21); benzene, dioxane, acetic acid (100:50:5) and ethyl acetate, formic acid (8O:l) (22); organic phase of ethyl acetate, isooctane, acetic acid, water (9:5:2:10) (23); benzene dioxane, acetic acid (40:40:2) (24); and chloroform, methanol, acetic acid, water (90:9:1:0.65) (25). Unlabelled standards were visualized and marked. The plates were scraped in 0.35 cm increments, and the radioactivity in each fraction was determined by liquid scintillation counting techniques. [3]H-6-Keto-PGFl was prepared by addition of [3]H-PG12 to distilled water, followe% by acidification and extraction, and the product was compared with authentic standards by TLC as described above. Lunq Homogenates Male rats (Charles River CD, purchased from Charles River, Wilmington, MA) weighing 200-3509 were anesthesized with halothane. Lungs were excised, cleaned, weighed, chopped and homogenized in 0.25M sucrose, pH - 7.0 with 0.5M Hepes buffer (tissue to sucrose ratio, 1:lO). The homogenate was centrifuged at 600 xg to remove tissue debris. A portion of the supernatant was boiled for 30 min to destroy the enzymes. Incubation mixtures consisted of 4.0 mM B-NAD, O.lM Tris buffer pH 8.5, and 2.5 ml boiled or nonboiled supernatant of lung homogenate in a total volume of 5.0 ml. After a 2 min preincubation at 37'C, [3]H-PG12, [3]H-6-keto-PGFl,or [3]H-PGF2, (dilution of labelled with unlabelled) was added to the incubation mixture to a final concentration of 10nM. The pH of the final incubation mixture was 8.5. This pH was chosen because PG12 is stable at pH > 8.4 (5) and the prostaglandin dehydrogenase exhibits optimal actiyity at pH - 8.0 (26). At timed intervals aliquots of the incubation mixture were added to tubes containing the appropriate amount of 0.5 N HCl to adjust the pH to 3.3, and the solutions were imnediately extracted and examined by TLC. Isolated Perfused Lungs Male rats weighing 200-3009 were anesthesized with halothane and surgically prepared for lung perfusion. Our IPL technique has been previously described in detail (18). The perfusion medium was Kreb's-Ringer buffer containing 5 mM glucose and 4.5% bovine serum albumin (BSA) or Kreb's-Ringer alone. The medium was adjusted to pH 7.8-8.0 with 10 M Tris base, maintained at that pH with 5% CO2 in oxygen and heated to 37'C. Perfusion success was determined by injection of blue dextran or India ink into the pulmonary artery via
DECEMBER 1978 VOL. 16 NO. 6
873
PROSTAGLANDINS
an injection port in the apparatus. Lack of color‘indicated nonperfused areas; lungs less than 90% perfused were discarded. All lungs showed < 10% increase in weight at the end of experimentation, indicating little or no edema formation. [3]H-PG (PG12, 6-keto-PGFl or PGF2,, 15 or 50 pmoles each) and, in some cases, the vascular marger [14]C-dextran (-90,000 dpm) were injected into the pulmonary artery of the IPL as a bolus. The perfusate flow rate was 30 ml/min and was monitored by a flow transducer. Effluent from the lung was collected via the pulmonary vein by means of a fraction collector at 1.14 set intervals for 50 set or as a single fraction over a total of 40 sec. [3]H and [14]C in each sample were determined by liquid scintillation counting. Samples collected at 1.14 set intervals were pooled, and aliquots were acidified, extracted and chromatographed as described above. RESULTS AND DISCUSSION One system responsible for the metabolism of circulating PGs consists of a carrier-mediateduptake of PGs from the pulmonary circulation to inside the cells (18) and intracellular enzymes which degrade the PGs (27). Dusting -et al. have studied the disappearance of PG12 in the circulation of the dog using bioassay techniques (16). Their work indicated little or no inactivation of PG12 on passage through the lung, while extensive inactivation occurred in the liver and hind quarters. Similarly, the conclusion that PG12 escapes pulmonary metabolism has been reached by Bolger and coworkers while studying the renal actions of PG12 (19), and by Armstrong --* et al in a study of hypotension induced by PG12 (13). To compare the capability of lung homogenates to metabolize PG12 and its non-enzymatic breakdown product vs. other more thoroughly investigated classical PGs, the metabolism of [3]H-PG12, [3]H-6-ketoPGFlc,and [3]H-PGF2, was examined. Incubation of rat lung homogenates with [3]H-PGF2, resulted in the formation of two distinct metabolite peaks which co-chromatographed with authentic 15-keto-PGF2, and 13,14-dihydro-15-keto-PGF2,. As found previously (18,28), the rat lung seems to lack the 15-ketoprostaglandin reductase that is necessary for formation of the 13,14dihydro-PG metabolites. Two chromatographic eaks resulted from the incubation of [3]HPGI2 (see Figure 1) or [3'; H-6-keto-PGFl, with rat lung homogenates. The peak remaining closest to the origin co-chromatographedwith 6-keto-PGFl,, the breakdown product of PG12, and 6-keto-13,14-dihydroPGFl . Since the rat lung seems incapable of forming the 13,14dihy%ro-PG metabolites, the initial peak probably only represents unchanged PG12 in the case of the PG12 incubation mixtures and unchanged 6-keto-PGFl, in mixtures starting with 6-keto-PGFl,. The peak closest to the solvent front could be either 6,15-diketo-PGFl, or 6,15-diketo-13,14-dihydro-PGFl, or a mixture of these metabolites. Further attempts to separate these metabolites by means of other TLC
874
DECEMBER 1978VOL.16NO.6
PROSTAGLANDINS
t
.*
Fig. 1.
0
4
7 Cm tmm origin
front
Thin Layer Chromatography of Extract from Incubation of [3]H-PG12 with Rat Lung Homogenates. [3]H-PGI2 was incubated with 600 xg supernatant of rat lung homogenates in the presence of B-NAD at 37"C, pH 8.4, for 10 min and extracted as described in METHODS. The extract and authentic standards were applied to silica gel G plates which were developed in chloroform, methanol, acetic acid, water (90:9:1:0.65).Unlabelled standards of 6-keto-PGFl and 6-keto-13,14-dihydro-PGFla(I), 6-15-diketo-PGFl, (I?) and 6,15-diketo-13,14-dihydro-PGFla(III) were used for identification purposes.
systems as listed in METHODS were unsuccessful. However, Wong --* et al (29) have recently used GCMS to isolate and identify 6,15-diketoPGFla as a major metabolite formed from incubation of PG12 with the cytoplasm of various tissues from rats and rabbits, including lung. The major metabolite formed in our incubation systems was, therefore, most likely 6,15-diketo-PGFl,. Since PG12 is relatively stable at pH 8.5, we conclude that the 6,15-diketo-PGFl,found in this incubation system probably arose from non-enzymatic hydrolysis of 15-ketoPG12 during the workup procedure. PG12 and PGF2c,appeared to be metabolized to comparable extents by rat lung homogenates over a 10 min incubation period (see Figure 2). After 4 min 16% of both the PGF2cland PG12 was degraded, while after 10 min, 41% of the PGF2, was metabolized compared to 35% of
DECEMBER 1978 VOL. 16 NO. 6
875
the PG12. In contrast, 6-keto-PGFl, was only minimally metabolized (6% at 10 min). Little or no metabolism & 2%) of any PG was obtained with boiled homogenates.
_o_-_,4---
0
Fig. 2.
2
_-o-“~ I
I
-Q
-
4 6 Time (min)
I
8
1
IO
Time Course for Metabolism of [3]H-PGF2,, [3]H-PG12 and [3]H-6-keto-PGFl,by Rat Lung Homogenates. 1OnM [3]H-PGF2, (A), [3]H-PG12 (0), or [J]H-6-keto-PGFl, (0) was incubated with 600 xg rat lung supernatant plus B-NAD at pH 8.5, and was extracted and chromatographed as described in METHODS.
Having shown that rat lung homogenates do degrade PG12, we investigated the effect of passage through the rat pulmonary circulation upon this PG and 6-keto-PGFla as compared to PGF2 . The effluent from the rat IPL was collected at 1.14 set intervals after bolus injections of 15 pmoles [S]H-PGF2,, together with the vascular marker [14]C-dextran, into the pulmonary artery. Analysis of the effluent for radioactivity showed a displaced tritium peak (Figure 3) as we have previously shown (18), indicating that PGF2 was taken up into the pulmonary vascular cells. TLC analysis revealed that 94% of the PGF2, was metabolized (see Table 1). 15-Keto-PGF2, and 13,14dihydro-15-veto-PGF2,were the metabolites formed. At a dose of 55 pmoles of [3]H-PGF2,, 63% of the tritium was detected as PGF2,metabolites.
876
DECEMBER 1978 VOL. 16 NO. 6
PROSTAGLANDINS
9
6;6 G t 8 3
I
Fig. 3.
IO
20 30 Sompl.no.
40
Appearance of [3]H (A) and [14]C (8) in the Venous Effluent of the Rat IPL after a Bolus Injection of [3]H-PGF2, and [14]C-Dextran. IPLs were perfused as described in METHODS using Kreb's-Ringer buffer plus 5mM lucose and 4.5% BSA. Injections of 15 pmoles [3sH-PGF2 and -90,000 dpm [14]C-dextran were made into tffepulmonary artery of the IPL and analyses were performed as described in METHODS. Samples were collected at 1.14 set intervals, and 0.2 ml aliquots were assayed for radioactivity.
DECEMBER 1978 VOL. 16 NO. 6
PROSTAGLANDINS
PG
Dose
pGF2cX
15 pmoles 54 pmoles
PG12
15 pmoles 48 pmoles
6-keto-PGFIU
15 pmoles 50 pmoles
Table 1.
% Metabolized 94 + 1 (N = 10) 95 f 1 (N = 4)
STUDIES OF THE MECHANISMS INVOLVED IN THE FATE OF PROSTACYCLIN (PGQ)
AND 6-KETO-PGF,a IN THE
PULMONARY CIRCULATION Hollis J. Hawkins,' J. Bryan Smith* Kyriacos C. Nicolaou,' and Thomas E. Eling' 1 Prostaglandins Section Laboratory of Pulmonary Function and Toxicology National Institute of Environmental Health Sciences Research Triangle Park, North Carolina 27709 *Cardeza Foundation Thomas Jefferson University Philadelphia, Pennsylvania 19107 'Department of Chemistry University of Pennsylvania Philadelphia, Pennsylvania 19104
ABSTRACT We have investigated the metabolism of [3]H_prostaglandin(PG)I2 and its non-enzymatic breakdown product [3]H-6-keto-PGFl,by rat pulmonary tissue and their possible uptake and metabolism upon passage through the isolated perfused rat lung. When incubated with rat lung homogenate in the presence of B-NAD, [3]H-PGI was extensively degraded into at least one metabolite, while [35H-6-keto-PGFl was only minimally metabolized. However, on passage through isolate% perfused rat lungs, neither [3]H-PG12 nor [3]H-6-keto-PGFl were removed from the circulation into the lung or degraded. Tiis demonstration that PG12 is not a substrate for the transport system for the removal of PGs from the circulation into the lung further illustrates that this system is a critical determinant for the pulmonary inactivation of circulating prostaglandins. The experimental findings are discussed in reference to the structure-activityrequirements necessary for pulmonary transport and subsequent metabolism.
DECEMBER 1978 VOL. 16 NO. 6
871
PROSTAGLANDINS
INTRODUCTION Prostacyclin, or prostaglandin (PG)Iz, has recently been shown to be a novel PG biosynthesized from the PG endoperoxides PGG2 and PGH2 (l-4). Studies involving the direct measurement of PG12 or the detection of its single non-enzymatic, biologically inactive breakdown product, 6-keto-PGFl, (5), have shown that PG12 is formed by arteries of various species, human veins, rat stomach fundus, and animal lungs, seminal vesicles and heart (l-4, 6-11). PG12 is the most potent of the PG inhibitors of platelet aggregation yet identified (l-3), and has been shown to be a powerful coronary vasodilator (12-14). The balance between levels of this bicyclic PG and thromboxane (TX) A2 appears to play a critical role in the control of thrombus formation (15). Since the half-life of PG12 in blood at physiological pH (16) is long enough to make it an important circulating hormone, its possible metabolism during passage through the circulation could affect the PGI2-TXA balance. It is well established that the fung plays a major role in the metabolism of circulating PGs (for review see ref. 17). We have previously demonstrated that a transport system is necessary for the passage of PGs into the pulmonary cells for subsequent inactivation by intracellular enzymes, and that the substrate specificity for the inactivation of circulating PGs appears to reside with the transport system (18). An apparent lack of biological inactivation of PG12 on passage through the lung has been suggested by several investigators (13,16, 19). In this paper we have directly studied the pulmonary metabolism of PG12 and 6-keto-PGFl and have investigated the underlying mechanisms responsible for &e lack of inactivation by (i) examining the metabolism of [3]H-PG12 and its hydrolysis product [SJH-6-keto-PGFl, by rat lung homogenates, and (ii) studying the passage of [3]H-PG12 and [3]H-6-keto-PGFl,through the isolated perfused rat lung. MATERIALS AND METHODS [9-(3)H]-PG12 (10.9 Ci/mmole or 12.0 Ci/mmole) was prepared according to the method of Nicolaou -_ et al. (20). [9-(3)-HI-PGF2, (9.2 Ci/mmole) and [14]C - dextran (M.W. - 70,000, 1.043 mCi/gram) were purchased from New England Nuclear, Boston, MA. Authentic standards of PGE2, PGF2,-THAM, 13,14-dihydro-PGFp,,13,14-dihydro15-keto-PGFp,, 15-keto-PGF2,, and 6-keto-PGFl, were gifts from Dr. John Pike, Upjohn Co., Kalamazoo, MI. Dr. Frank Sun of Upjohn CO. kindly provided authentic standards of 6-keto-13,14-dihydro-PGFl., 6,15-diketo-13,14-dihydro-PGFl. and 6,15-diketo-PGFl,. Silica gel G thin layer plates (250~) were purchased from Analtech Inc., Newark, DE. B-NAD was obtained from Sigma Chemical Co., St. Louis, MO. Blue dextran (M.W. -.2 X 106) was purchased from Pharmacia Fine Chemicals, Piscataway, NJ. Lung homogenate incubation mixtures and isolated perfused lung
872
DECEMBER 1978 VOL. 16 NO. 6
PROSTAGLANDINS
(IPL) effluents were analyzed for metabolites by thin layer chromatography (TLC). Samples were acidified to pH 3.0-3.5 with HCl and extracted twice with 6 vol ethyl acetate or ether, and the extracts were evaporated to dryness in vacua. The residues were dissolved in methanol or acetone and ali?$ioEand unlabelled standards were applied to silica gel plates which were developed in chloroform, methanol, acetic acid, water (135:12:1.5:1.2)(21); benzene, dioxane, acetic acid (100:50:5) and ethyl acetate, formic acid (8O:l) (22); organic phase of ethyl acetate, isooctane, acetic acid, water (9:5:2:10) (23); benzene dioxane, acetic acid (40:40:2) (24); and chloroform, methanol, acetic acid, water (90:9:1:0.65) (25). Unlabelled standards were visualized and marked. The plates were scraped in 0.35 cm increments, and the radioactivity in each fraction was determined by liquid scintillation counting techniques. [3]H-6-Keto-PGFl was prepared by addition of [3]H-PG12 to distilled water, followe% by acidification and extraction, and the product was compared with authentic standards by TLC as described above. Lunq Homogenates Male rats (Charles River CD, purchased from Charles River, Wilmington, MA) weighing 200-3509 were anesthesized with halothane. Lungs were excised, cleaned, weighed, chopped and homogenized in 0.25M sucrose, pH - 7.0 with 0.5M Hepes buffer (tissue to sucrose ratio, 1:lO). The homogenate was centrifuged at 600 xg to remove tissue debris. A portion of the supernatant was boiled for 30 min to destroy the enzymes. Incubation mixtures consisted of 4.0 mM B-NAD, O.lM Tris buffer pH 8.5, and 2.5 ml boiled or nonboiled supernatant of lung homogenate in a total volume of 5.0 ml. After a 2 min preincubation at 37'C, [3]H-PG12, [3]H-6-keto-PGFl,or [3]H-PGF2, (dilution of labelled with unlabelled) was added to the incubation mixture to a final concentration of 10nM. The pH of the final incubation mixture was 8.5. This pH was chosen because PG12 is stable at pH > 8.4 (5) and the prostaglandin dehydrogenase exhibits optimal actiyity at pH - 8.0 (26). At timed intervals aliquots of the incubation mixture were added to tubes containing the appropriate amount of 0.5 N HCl to adjust the pH to 3.3, and the solutions were imnediately extracted and examined by TLC. Isolated Perfused Lungs Male rats weighing 200-3009 were anesthesized with halothane and surgically prepared for lung perfusion. Our IPL technique has been previously described in detail (18). The perfusion medium was Kreb's-Ringer buffer containing 5 mM glucose and 4.5% bovine serum albumin (BSA) or Kreb's-Ringer alone. The medium was adjusted to pH 7.8-8.0 with 10 M Tris base, maintained at that pH with 5% CO2 in oxygen and heated to 37'C. Perfusion success was determined by injection of blue dextran or India ink into the pulmonary artery via
DECEMBER 1978 VOL. 16 NO. 6
873
PROSTAGLANDINS
an injection port in the apparatus. Lack of color‘indicated nonperfused areas; lungs less than 90% perfused were discarded. All lungs showed < 10% increase in weight at the end of experimentation, indicating little or no edema formation. [3]H-PG (PG12, 6-keto-PGFl or PGF2,, 15 or 50 pmoles each) and, in some cases, the vascular marger [14]C-dextran (-90,000 dpm) were injected into the pulmonary artery of the IPL as a bolus. The perfusate flow rate was 30 ml/min and was monitored by a flow transducer. Effluent from the lung was collected via the pulmonary vein by means of a fraction collector at 1.14 set intervals for 50 set or as a single fraction over a total of 40 sec. [3]H and [14]C in each sample were determined by liquid scintillation counting. Samples collected at 1.14 set intervals were pooled, and aliquots were acidified, extracted and chromatographed as described above. RESULTS AND DISCUSSION One system responsible for the metabolism of circulating PGs consists of a carrier-mediateduptake of PGs from the pulmonary circulation to inside the cells (18) and intracellular enzymes which degrade the PGs (27). Dusting -et al. have studied the disappearance of PG12 in the circulation of the dog using bioassay techniques (16). Their work indicated little or no inactivation of PG12 on passage through the lung, while extensive inactivation occurred in the liver and hind quarters. Similarly, the conclusion that PG12 escapes pulmonary metabolism has been reached by Bolger and coworkers while studying the renal actions of PG12 (19), and by Armstrong --* et al in a study of hypotension induced by PG12 (13). To compare the capability of lung homogenates to metabolize PG12 and its non-enzymatic breakdown product vs. other more thoroughly investigated classical PGs, the metabolism of [3]H-PG12, [3]H-6-ketoPGFlc,and [3]H-PGF2, was examined. Incubation of rat lung homogenates with [3]H-PGF2, resulted in the formation of two distinct metabolite peaks which co-chromatographed with authentic 15-keto-PGF2, and 13,14-dihydro-15-keto-PGF2,. As found previously (18,28), the rat lung seems to lack the 15-ketoprostaglandin reductase that is necessary for formation of the 13,14dihydro-PG metabolites. Two chromatographic eaks resulted from the incubation of [3]HPGI2 (see Figure 1) or [3'; H-6-keto-PGFl, with rat lung homogenates. The peak remaining closest to the origin co-chromatographedwith 6-keto-PGFl,, the breakdown product of PG12, and 6-keto-13,14-dihydroPGFl . Since the rat lung seems incapable of forming the 13,14dihy%ro-PG metabolites, the initial peak probably only represents unchanged PG12 in the case of the PG12 incubation mixtures and unchanged 6-keto-PGFl, in mixtures starting with 6-keto-PGFl,. The peak closest to the solvent front could be either 6,15-diketo-PGFl, or 6,15-diketo-13,14-dihydro-PGFl, or a mixture of these metabolites. Further attempts to separate these metabolites by means of other TLC
874
DECEMBER 1978VOL.16NO.6
PROSTAGLANDINS
t
.*
Fig. 1.
0
4
7 Cm tmm origin
front
Thin Layer Chromatography of Extract from Incubation of [3]H-PG12 with Rat Lung Homogenates. [3]H-PGI2 was incubated with 600 xg supernatant of rat lung homogenates in the presence of B-NAD at 37"C, pH 8.4, for 10 min and extracted as described in METHODS. The extract and authentic standards were applied to silica gel G plates which were developed in chloroform, methanol, acetic acid, water (90:9:1:0.65).Unlabelled standards of 6-keto-PGFl and 6-keto-13,14-dihydro-PGFla(I), 6-15-diketo-PGFl, (I?) and 6,15-diketo-13,14-dihydro-PGFla(III) were used for identification purposes.
systems as listed in METHODS were unsuccessful. However, Wong --* et al (29) have recently used GCMS to isolate and identify 6,15-diketoPGFla as a major metabolite formed from incubation of PG12 with the cytoplasm of various tissues from rats and rabbits, including lung. The major metabolite formed in our incubation systems was, therefore, most likely 6,15-diketo-PGFl,. Since PG12 is relatively stable at pH 8.5, we conclude that the 6,15-diketo-PGFl,found in this incubation system probably arose from non-enzymatic hydrolysis of 15-ketoPG12 during the workup procedure. PG12 and PGF2c,appeared to be metabolized to comparable extents by rat lung homogenates over a 10 min incubation period (see Figure 2). After 4 min 16% of both the PGF2cland PG12 was degraded, while after 10 min, 41% of the PGF2, was metabolized compared to 35% of
DECEMBER 1978 VOL. 16 NO. 6
875
the PG12. In contrast, 6-keto-PGFl, was only minimally metabolized (6% at 10 min). Little or no metabolism & 2%) of any PG was obtained with boiled homogenates.
_o_-_,4---
0
Fig. 2.
2
_-o-“~ I
I
-Q
-
4 6 Time (min)
I
8
1
IO
Time Course for Metabolism of [3]H-PGF2,, [3]H-PG12 and [3]H-6-keto-PGFl,by Rat Lung Homogenates. 1OnM [3]H-PGF2, (A), [3]H-PG12 (0), or [J]H-6-keto-PGFl, (0) was incubated with 600 xg rat lung supernatant plus B-NAD at pH 8.5, and was extracted and chromatographed as described in METHODS.
Having shown that rat lung homogenates do degrade PG12, we investigated the effect of passage through the rat pulmonary circulation upon this PG and 6-keto-PGFla as compared to PGF2 . The effluent from the rat IPL was collected at 1.14 set intervals after bolus injections of 15 pmoles [S]H-PGF2,, together with the vascular marker [14]C-dextran, into the pulmonary artery. Analysis of the effluent for radioactivity showed a displaced tritium peak (Figure 3) as we have previously shown (18), indicating that PGF2 was taken up into the pulmonary vascular cells. TLC analysis revealed that 94% of the PGF2, was metabolized (see Table 1). 15-Keto-PGF2, and 13,14dihydro-15-veto-PGF2,were the metabolites formed. At a dose of 55 pmoles of [3]H-PGF2,, 63% of the tritium was detected as PGF2,metabolites.
876
DECEMBER 1978 VOL. 16 NO. 6
PROSTAGLANDINS
9
6;6 G t 8 3
I
Fig. 3.
IO
20 30 Sompl.no.
40
Appearance of [3]H (A) and [14]C (8) in the Venous Effluent of the Rat IPL after a Bolus Injection of [3]H-PGF2, and [14]C-Dextran. IPLs were perfused as described in METHODS using Kreb's-Ringer buffer plus 5mM lucose and 4.5% BSA. Injections of 15 pmoles [3sH-PGF2 and -90,000 dpm [14]C-dextran were made into tffepulmonary artery of the IPL and analyses were performed as described in METHODS. Samples were collected at 1.14 set intervals, and 0.2 ml aliquots were assayed for radioactivity.
DECEMBER 1978 VOL. 16 NO. 6
PROSTAGLANDINS
PG
Dose
pGF2cX
15 pmoles 54 pmoles
PG12
15 pmoles 48 pmoles
6-keto-PGFIU
15 pmoles 50 pmoles
Table 1.
% Metabolized 94 + 1 (N = 10) 95 f 1 (N = 4)
