Pulmonary Pharmacology (1990) 3 73-77 ©1990 Longman Group UK Ltd
0952-0600/90/0003-0073/$10 .00
PULMONARY PHARMACOLOGY
Treatment in vivo with Ph CL28A alters Prostaglandin E 2 , Prostacyclin and Leukotriene C 4 Metabolism in Rat Isolated Lung J . J . Pankhania, Y . S . Bakhle* Department of Pharmacology, Hunterian Institute, Royal College of Surgeons, Lincoln's Inn Fields, London SUMMARY. We have studied the effects of a potent inhibitor of prostaglandin (PG) catabolism, Ph CL28A, given in vivo, on PG metabolism in rat perfused lungs, isolated at different times after treatment . Two doses of Ph CL28A were used, 10 and 30 mg/kg, and lungs isolated at 1, 2 and 4h after a single injection (i .p.). The catabolism of exogenous PGE2 was decreased for up to 2h after injection . Synthesis of PGI 2 using exogenous arachidonic acid, was increased by treatment with Ph CL28A . Stimulation of the turnover of endogenous arachidonic acid with the calcium ionophore A23187 led to the synthesis of LTC 4 as well as PGI 2 . Treatment in vivo with Ph CL28A, decreased the output of LTC 4 but increased that of PGI 2 . However in the presence of indomethacin, the perfused lung did not form any PGI 2 and the output of LTC 4 was still inhibited by Ph CL28A . We conclude that the inhibition of LT formation in lung by Ph CL28A was not due to the increased production of PGI 2 . These two properties, inhibition of LT synthesis and potentiation of PGI 2 formation, may synergize in vivo to give antiinflammatory activity.
INTRODUCTION
solution (6 mg/ml) freshly made in 0 .5% (w/v) Na2CO 3 ) or an equal volume of the Na2 CO 3 solution only (sham treated) . At fixed times after this injection, rats were anaesthetized with pentobarbitone (60 mg/kg ; i .p .), lungs were removed and perfused via the pulmonary artery with Krebs solution (gassed with 95% 02 ; 5% CO 2 and warmed to 37 0) at 8 ml/min .' When PG inactivation was studied, the Krebs solution contained indomethacin (5µg/ml) . After a 10 min period of perfusion the assays were started .
The azobenzene compound Ph CL28A is a potent inhibitor of prostaglandin 15-hydroxydehydrogenase (PGDH), the major enzyme in prostaglandin inactivation .' This compound, perfused through isolated lungs, also increased the output of PGI 2 following stimulation with the calcium ionophore A23187 . 2 More recently, inhibition of leukotriene (LT) synthesis in vitro has been demonstrated with Ph CL28A . 3 Synthesis of LTs can be decreased by prostaglandins 4-6 and one mechanism by which LT synthesis might be affected by Ph CL28A is via an increase in the amounts of PGs synthesized concomitantly . Synthesis of LTs is an important property of lung' and we have investigated the possible interactions between PGI 2 and LT synthesis in lungs isolated from rats treated with Ph CL28A in vivo . A preliminary account of some of these results has been given to the British Pharmacological Society .'
Efflux of radioactivity - T1/2 assay Here the effluent from rat lungs was collected in 4drop fractions (each approximately 3 s) immediately before and after an injection of 14C-PGE2 (500 ng . 0 .01 µCi, 0 .1 ml) into the Krebs perfusate entering the lung . The total radioactivity in each fraction was measured by liquid scintillation methods . Catabolism and output of eicosanoids
MATERIALS AND METHODS
The survival of exogenous PGE 2 was measured by giving a single bolus injection of 500 ng of PGE 2 , into the perfusate entering the lung . The effluent perfusate was collected in a single fraction for 5 min subsequently and a sample of this effluent taken for RIA of unchanged PGE 2 . Output of PGs from exogenous AA was measured following an injection of 10 µg AA and collection of lung effluent for 5 min . To study output from AA in
Preparation of isolated lungs Male Wistar rats (200-250 g) were used in three groups ; untreated, drug treated and sham treated . The last two groups received either Ph CL28A by intraperitoneal injection (10 or 30 mg/kg body weight of a *To whom correspondence should be addressed 73
74
Pulmonary Pharmacology
lung lipids (endogenous AA), the calcium ionophore A23187 (3 µg) was injected into the perfusate entering the lung . The lung effluent was collected for 5 min immediately following the ionophore . In both series of experiments, immediately before the stimulus (AA or ionophore), a 5 min collection of lung effluent was made to provide samples of the basal output of eicosanoids . Radioimmunoassay (RIA) Two methods were used . After exogenous PGE 2 or AA, the lung effluent was assayed without extraction . A sample of lung effluent (0 .1 ml) was assayed directly or after five-fold dilution with Tris-buffered saline, for PGE 2 or 6-oxo-PGF,,, . After ionophore stimulation, the levels of the eicosanoids in lung effluent were too low to allow direct assay of the effluent . They were concentrated by passing the acidified (pH3-3 .5) effluent through a cartridge of Sep-Pak C 18 at a flow rate of 5 ml/min to adsorb the eicosanoids . The cartridge was washed with water (10 ml) and the eicosanoids eluted with methanol (5 ml) . The methanol was evaporated under reduced pressure and the residue stored under nitrogen at - 20°C until the RIA was carried out . Recoveries of LTC 4 and 6-oxo-PGF,,, were at least 80% . No corrections have been made for recovery in the results presented . The RIA procedures have been described previously. 1 o, I 1 Cross-reactivities of the anti-sera were as follows : anti-PGE 2; 6-oxo-PGF 1a =0.04%, PGF2a = 1 .4%, 15-oxo-PGE 2 = 1 .2%, 13,14-dihydro15-oxo-PGE 2 =0.6% : anti-6-oxo-PGF,,, ; PGE 2 = 4.8%, PGF 2a =5.4%, 15-oxo derivatives of these PGs=0 .5% : anti-LTC 4; LTB 4 =0.05%, LTD,= 29 .4%, LTE 4 =0.7% . Because there was relatively extensive cross-reaction between LTC 4 and LTD 4, we have referred to the LTs assayed here as 'immunoreactive LTC 4' ( ir-LTC4). The lower limits of detection of the RIA were for PGE 2, 0.2 ng/ml, 6-oxoPGF, a, 0.1 ng/ml and LTC 4, 2 .5 ng/ml . Materials Arachidonic acid (Sigma, Poole) was dissolved in ethanol and stored under nitrogen at - 20°C . After evaporating the ethanol with a stream of nitrogen, the residue was taken up in 0 .9% w/v NaCl solution and converted to the sodium salt of AA with Na 2CO 3. This aqueous solution was freshly made for each day's experiments . Prostaglandin E 2 (Sigma, Poole) was stored in methanol solution at - 20 °C. Methanol was evaporated and the PG redissolved in 0 .9% w/v NaCl solution and diluted further to the desired concentration . 5, 8, 9, 11, 12, 14, 15 (n)_ 3 H-6-oxo-PGF,,,, (100 Ci/nmol) and 14,15 (n)- 3 H-LTC4 (36 Ci/mmol) were
obtained from New England Nuclear, Boston, Mass . USA . 5, 6, 8, 11, 12, 14, 15, (n)- 3 H-PGE 2 (120 Ci/mmol) for the RIA and 1- 14 C PGE 2 (58mCi/mmol) for the T1/2 assays were obtained from Amersham International (Amersham, Bucks) . The calcium ionophore was generously provided by Dr W . Dawson (Lilly Research Centre, Windlesham, Surrey) and indomethacin by Merck Sharp and Dohme (Hoddesdon, Herts) . We are also grateful for a gift of Ph CL28A from Mr H . Agback (Pharmacia, Uppsala, Sweden) . The antiserum to 6-oxo-PGF,,, was kindly donated by Dr J . A . Salmon (Wellcome Foundation, Beckenham, Kent) and the antisera to PGE2 and LTC 4 were prepared in this laboratory . Statistical methods Results are given as mean values (±s .e . mean) from N lungs unless otherwise stated . The unpaired t-test was used to assess significance of the difference between means and a value of P < 0 .05 was taken to denote a significant difference .
RESULTS Catabolism of exogenous PGE 2 Our first experiments were to establish the times and doses required to show that a property of Ph CL28A, given by continuous infusion, i.e ., inhibition of PGE 2 inactivation in lung, was also exhibited in the isolated lung after treatment in vivo . As seen in Figure 1, the catabolism of exogenous PGE 2 was decreased, i .e ., survival was increased, for at least 2h after treatment with either dose of Ph CL28A . The higher dose of Ph CL28A (30 mg/kg) showed a trend towards a higher survival but, over the doses used here, the dose dependency of this response was not striking . As noted earlier with continuous infusions of Ph CL28A, 2 the increased survival of PGE 2 after in vivo treatment was also accompanied by an increase in T1/2 measured with 14 C-PGE 2, to 2-3 times the normal values (lower half of Fig . 1) . Sham treatment did not affect either the survival or the T1/2 values . Output of 6-oxo-PGF Ia following exogenous AA In initial experiments, output of 6-oxo-PGF, . was measured in lungs taken at 2h after Ph CL28A treatment . This time was close to that used in previous work 12 and PGE 2 survival was still 3-6 times the normal values . There was no effect on 6-oxo-PGF Ia output under these conditions (3 .3 ±0.4 ng/ml effluent after treatment vs . 3.1 ± 0 .4 ng/ml untreated, N = 4) and we therefore utilized the lungs at I h after treatment . At this time, as shown in Figure 2, output of 6oxo-PGF Ia following the bolus of exogenous AA was
Ph CL28A and Eicosanoid Metabolism in Lung
75
100 O 10 mg/kg 3
S w 0 t N W 0 a w 0
30 mg/kg
80 60 40
0 20
ni
0
4
hours
Output of PGI2 from rat isolated lung following exogenous AA ; effect of Ph CL28A given in vivo . Two levels of Ph CL28A were used (10 and 30 mg/kg) and the lungs taken only at lh after treatment . The basal output of PGI 2 , assayed as 6oxo-PGF,,, increased dose dependently with Ph CL28A treatment but the output after exogenous AA was maximally increased at 10 mg/kg . The values shown are the mean (± s .e .mean) results from 4-5 lungs in each condition; significant differences from the relevant untreated values are marked*, P < 0 .05 . Fig. 2 -
0--0
100
- • 30 mg/kg
#
v
10mg/kg
80
0 60
N H
40
12-
O untreated ifff~ CL28A
0 20 U
1
2
4
hours
Fig. I - Effect of treatment with Ph CL28A in vivo on PGE 2 pharmacokinetics in rat isolated lung . Two levels of Ph CL28A were used (10 and 30 mg/kg) and the lungs taken at times up to 4h after treatment . In the upper half of the Figure, the survival of a test injection of PGE 2 was about 10% in untreated rats (U) but increased strikingly 1-2h after Ph CL28A . By 4h, the effects of Ph CL28A had been dissipated . In the lower half of the Figure, over the same time course, the efflux of 14C from the lung following 14 C-PGE 2 injection was much slower, i .e ., longer T1/2, shortly after Ph CL28A and this effect also had largely disappeared by 4h . The values shown are the mean (±s .e .mean) results from 4-5 lungs in each condition; significant differences from the relevant untreated values are marked*, P
0952-0600/90/0003-0073/$10 .00
PULMONARY PHARMACOLOGY
Treatment in vivo with Ph CL28A alters Prostaglandin E 2 , Prostacyclin and Leukotriene C 4 Metabolism in Rat Isolated Lung J . J . Pankhania, Y . S . Bakhle* Department of Pharmacology, Hunterian Institute, Royal College of Surgeons, Lincoln's Inn Fields, London SUMMARY. We have studied the effects of a potent inhibitor of prostaglandin (PG) catabolism, Ph CL28A, given in vivo, on PG metabolism in rat perfused lungs, isolated at different times after treatment . Two doses of Ph CL28A were used, 10 and 30 mg/kg, and lungs isolated at 1, 2 and 4h after a single injection (i .p.). The catabolism of exogenous PGE2 was decreased for up to 2h after injection . Synthesis of PGI 2 using exogenous arachidonic acid, was increased by treatment with Ph CL28A . Stimulation of the turnover of endogenous arachidonic acid with the calcium ionophore A23187 led to the synthesis of LTC 4 as well as PGI 2 . Treatment in vivo with Ph CL28A, decreased the output of LTC 4 but increased that of PGI 2 . However in the presence of indomethacin, the perfused lung did not form any PGI 2 and the output of LTC 4 was still inhibited by Ph CL28A . We conclude that the inhibition of LT formation in lung by Ph CL28A was not due to the increased production of PGI 2 . These two properties, inhibition of LT synthesis and potentiation of PGI 2 formation, may synergize in vivo to give antiinflammatory activity.
INTRODUCTION
solution (6 mg/ml) freshly made in 0 .5% (w/v) Na2CO 3 ) or an equal volume of the Na2 CO 3 solution only (sham treated) . At fixed times after this injection, rats were anaesthetized with pentobarbitone (60 mg/kg ; i .p .), lungs were removed and perfused via the pulmonary artery with Krebs solution (gassed with 95% 02 ; 5% CO 2 and warmed to 37 0) at 8 ml/min .' When PG inactivation was studied, the Krebs solution contained indomethacin (5µg/ml) . After a 10 min period of perfusion the assays were started .
The azobenzene compound Ph CL28A is a potent inhibitor of prostaglandin 15-hydroxydehydrogenase (PGDH), the major enzyme in prostaglandin inactivation .' This compound, perfused through isolated lungs, also increased the output of PGI 2 following stimulation with the calcium ionophore A23187 . 2 More recently, inhibition of leukotriene (LT) synthesis in vitro has been demonstrated with Ph CL28A . 3 Synthesis of LTs can be decreased by prostaglandins 4-6 and one mechanism by which LT synthesis might be affected by Ph CL28A is via an increase in the amounts of PGs synthesized concomitantly . Synthesis of LTs is an important property of lung' and we have investigated the possible interactions between PGI 2 and LT synthesis in lungs isolated from rats treated with Ph CL28A in vivo . A preliminary account of some of these results has been given to the British Pharmacological Society .'
Efflux of radioactivity - T1/2 assay Here the effluent from rat lungs was collected in 4drop fractions (each approximately 3 s) immediately before and after an injection of 14C-PGE2 (500 ng . 0 .01 µCi, 0 .1 ml) into the Krebs perfusate entering the lung . The total radioactivity in each fraction was measured by liquid scintillation methods . Catabolism and output of eicosanoids
MATERIALS AND METHODS
The survival of exogenous PGE 2 was measured by giving a single bolus injection of 500 ng of PGE 2 , into the perfusate entering the lung . The effluent perfusate was collected in a single fraction for 5 min subsequently and a sample of this effluent taken for RIA of unchanged PGE 2 . Output of PGs from exogenous AA was measured following an injection of 10 µg AA and collection of lung effluent for 5 min . To study output from AA in
Preparation of isolated lungs Male Wistar rats (200-250 g) were used in three groups ; untreated, drug treated and sham treated . The last two groups received either Ph CL28A by intraperitoneal injection (10 or 30 mg/kg body weight of a *To whom correspondence should be addressed 73
74
Pulmonary Pharmacology
lung lipids (endogenous AA), the calcium ionophore A23187 (3 µg) was injected into the perfusate entering the lung . The lung effluent was collected for 5 min immediately following the ionophore . In both series of experiments, immediately before the stimulus (AA or ionophore), a 5 min collection of lung effluent was made to provide samples of the basal output of eicosanoids . Radioimmunoassay (RIA) Two methods were used . After exogenous PGE 2 or AA, the lung effluent was assayed without extraction . A sample of lung effluent (0 .1 ml) was assayed directly or after five-fold dilution with Tris-buffered saline, for PGE 2 or 6-oxo-PGF,,, . After ionophore stimulation, the levels of the eicosanoids in lung effluent were too low to allow direct assay of the effluent . They were concentrated by passing the acidified (pH3-3 .5) effluent through a cartridge of Sep-Pak C 18 at a flow rate of 5 ml/min to adsorb the eicosanoids . The cartridge was washed with water (10 ml) and the eicosanoids eluted with methanol (5 ml) . The methanol was evaporated under reduced pressure and the residue stored under nitrogen at - 20°C until the RIA was carried out . Recoveries of LTC 4 and 6-oxo-PGF,,, were at least 80% . No corrections have been made for recovery in the results presented . The RIA procedures have been described previously. 1 o, I 1 Cross-reactivities of the anti-sera were as follows : anti-PGE 2; 6-oxo-PGF 1a =0.04%, PGF2a = 1 .4%, 15-oxo-PGE 2 = 1 .2%, 13,14-dihydro15-oxo-PGE 2 =0.6% : anti-6-oxo-PGF,,, ; PGE 2 = 4.8%, PGF 2a =5.4%, 15-oxo derivatives of these PGs=0 .5% : anti-LTC 4; LTB 4 =0.05%, LTD,= 29 .4%, LTE 4 =0.7% . Because there was relatively extensive cross-reaction between LTC 4 and LTD 4, we have referred to the LTs assayed here as 'immunoreactive LTC 4' ( ir-LTC4). The lower limits of detection of the RIA were for PGE 2, 0.2 ng/ml, 6-oxoPGF, a, 0.1 ng/ml and LTC 4, 2 .5 ng/ml . Materials Arachidonic acid (Sigma, Poole) was dissolved in ethanol and stored under nitrogen at - 20°C . After evaporating the ethanol with a stream of nitrogen, the residue was taken up in 0 .9% w/v NaCl solution and converted to the sodium salt of AA with Na 2CO 3. This aqueous solution was freshly made for each day's experiments . Prostaglandin E 2 (Sigma, Poole) was stored in methanol solution at - 20 °C. Methanol was evaporated and the PG redissolved in 0 .9% w/v NaCl solution and diluted further to the desired concentration . 5, 8, 9, 11, 12, 14, 15 (n)_ 3 H-6-oxo-PGF,,,, (100 Ci/nmol) and 14,15 (n)- 3 H-LTC4 (36 Ci/mmol) were
obtained from New England Nuclear, Boston, Mass . USA . 5, 6, 8, 11, 12, 14, 15, (n)- 3 H-PGE 2 (120 Ci/mmol) for the RIA and 1- 14 C PGE 2 (58mCi/mmol) for the T1/2 assays were obtained from Amersham International (Amersham, Bucks) . The calcium ionophore was generously provided by Dr W . Dawson (Lilly Research Centre, Windlesham, Surrey) and indomethacin by Merck Sharp and Dohme (Hoddesdon, Herts) . We are also grateful for a gift of Ph CL28A from Mr H . Agback (Pharmacia, Uppsala, Sweden) . The antiserum to 6-oxo-PGF,,, was kindly donated by Dr J . A . Salmon (Wellcome Foundation, Beckenham, Kent) and the antisera to PGE2 and LTC 4 were prepared in this laboratory . Statistical methods Results are given as mean values (±s .e . mean) from N lungs unless otherwise stated . The unpaired t-test was used to assess significance of the difference between means and a value of P < 0 .05 was taken to denote a significant difference .
RESULTS Catabolism of exogenous PGE 2 Our first experiments were to establish the times and doses required to show that a property of Ph CL28A, given by continuous infusion, i.e ., inhibition of PGE 2 inactivation in lung, was also exhibited in the isolated lung after treatment in vivo . As seen in Figure 1, the catabolism of exogenous PGE 2 was decreased, i .e ., survival was increased, for at least 2h after treatment with either dose of Ph CL28A . The higher dose of Ph CL28A (30 mg/kg) showed a trend towards a higher survival but, over the doses used here, the dose dependency of this response was not striking . As noted earlier with continuous infusions of Ph CL28A, 2 the increased survival of PGE 2 after in vivo treatment was also accompanied by an increase in T1/2 measured with 14 C-PGE 2, to 2-3 times the normal values (lower half of Fig . 1) . Sham treatment did not affect either the survival or the T1/2 values . Output of 6-oxo-PGF Ia following exogenous AA In initial experiments, output of 6-oxo-PGF, . was measured in lungs taken at 2h after Ph CL28A treatment . This time was close to that used in previous work 12 and PGE 2 survival was still 3-6 times the normal values . There was no effect on 6-oxo-PGF Ia output under these conditions (3 .3 ±0.4 ng/ml effluent after treatment vs . 3.1 ± 0 .4 ng/ml untreated, N = 4) and we therefore utilized the lungs at I h after treatment . At this time, as shown in Figure 2, output of 6oxo-PGF Ia following the bolus of exogenous AA was
Ph CL28A and Eicosanoid Metabolism in Lung
75
100 O 10 mg/kg 3
S w 0 t N W 0 a w 0
30 mg/kg
80 60 40
0 20
ni
0
4
hours
Output of PGI2 from rat isolated lung following exogenous AA ; effect of Ph CL28A given in vivo . Two levels of Ph CL28A were used (10 and 30 mg/kg) and the lungs taken only at lh after treatment . The basal output of PGI 2 , assayed as 6oxo-PGF,,, increased dose dependently with Ph CL28A treatment but the output after exogenous AA was maximally increased at 10 mg/kg . The values shown are the mean (± s .e .mean) results from 4-5 lungs in each condition; significant differences from the relevant untreated values are marked*, P < 0 .05 . Fig. 2 -
0--0
100
- • 30 mg/kg
#
v
10mg/kg
80
0 60
N H
40
12-
O untreated ifff~ CL28A
0 20 U
1
2
4
hours
Fig. I - Effect of treatment with Ph CL28A in vivo on PGE 2 pharmacokinetics in rat isolated lung . Two levels of Ph CL28A were used (10 and 30 mg/kg) and the lungs taken at times up to 4h after treatment . In the upper half of the Figure, the survival of a test injection of PGE 2 was about 10% in untreated rats (U) but increased strikingly 1-2h after Ph CL28A . By 4h, the effects of Ph CL28A had been dissipated . In the lower half of the Figure, over the same time course, the efflux of 14C from the lung following 14 C-PGE 2 injection was much slower, i .e ., longer T1/2, shortly after Ph CL28A and this effect also had largely disappeared by 4h . The values shown are the mean (±s .e .mean) results from 4-5 lungs in each condition; significant differences from the relevant untreated values are marked*, P
