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Prostaglandin 15-hydroxydehydrogenase activity during pregnancy in rabbits and rats
European Journal of Pharmacology, 33 (1975) 405--408 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
Short communication PROSTAGLANDIN 15-HYDROXYDEHYDROGENASE ACTIVITY DURING PREGNANCY IN RABBITS AND RATS JULIAN M. EGERTON-VERNON* and JOHN R. BEDWANI**
Department of Pharmacology, University of Cambridg e, Medical School, Hills Road, Cambridge CB2 2QD, U.K. Received 26 June 1975, accepted 24 July 1975
J.M. EGERTON-VERNON and J.R. BEDWANI, Prostaglandin 15-hydroxydehydrogenase activity during pregnancy in rabbits and rats, European J. Pharmacol. 33 (1975) 405--408. Prostaglandin 15-hydroxydehydrogenase (PGDH) activity in the rabbit lung increased strikingly during pregnancy. This change did not occur in the kidney, or spleen. In the corresponding organs from the rat, PGDH activity was essentially the same in pregnant and non-pregnant animals. These results are discussed in relation to the hypothesis that the increased PGDH activity in the pregnant rabbit lung is secondary to an elevated concentration of prostaglandins in the venous blood. Prostaglandin El
Prostaglandin metabolism
1. Introduction The ability of the rabbit lung to inactivate prostaglandin E2 increases markedly during pregnancy (Bedwani and Marley, 1974). This change is associated with a large increase in NAD +- dependent prostaglandin 15-hydroxydehydrogenase (PGDH) activity, and a much smaller increase in prostaglandin A13-reductase activity (Sun and Armour, 1974 ,)The factor responsible for this is not known. Bedwani and Marley (1975) found that chronic treatment of non-pregnant rabbits with progesterone was followed by an increase in lung prostaglandin-metabolizing activity, but thought it unlikely that there is a direct relationship between plasma progesterone levels and prostaglandin metabolism in this organ. * Present address: Middlesex Hospital Medical School, Mortimer Street, London W1, U.K. ** Present address: Department" of Physiology, University College, P.O. Box 78, Cardiff CF1 l X L U.K. To whom requests for reprints should be sent.
Pregnancy
The present experiments were carried out to see whether an increased PGDH activity during pregnancy is confined to the lung, or whether it also occurs in other organs. Accordingly, we investigated the activity of this enzyme in lung, kidney, spleen and liver from pregnant and non-pregnant rabbits. For comparative purposes, similar experiments were also performed in the rat.
2. Materials and methods
Animals used were New Zealand White rabbits and Wistar rats. With the latter species, organs from 2 to 3 animals were pooled for each experiment. Non-pregnant rats were used when in dioestrus. Pregnant animals were sacrificed on day 19 (rats) or day 26 (rabbits) of gestation. PGDH activity was assayed by measuring the formation of 15-ketoprostaglandin E1 on incubation of cytoplasmic fractions from organ homogenates with prostaglandin E1 (PGE1)
406 and NAD *. The procedure was based on the method of .~,ngg~d et al. (1971). Organs were homogenized in 4 volumes of ice-cold buffer of the following composition (mM): K:HPO4 78.3; KH2 PO4 21.7; EDTA 1.0; mercaptoethanol 8.0. A high speed supernatant was prepared from each homogenate by centrifugation at 1400 g for 3 min, then at 100,000 g for I hr (at 4 °C). Aliquots of the supernatant were taken for protein assay (Goa, 1953), and to 5 ml of the remainder were added NAD ÷, sodium pyruvate and lactate dehydrogenase to give final concentrations of 10 mM, 50 mM and 50 pg/ml respectively. The sodium pyruvate and lactate dehydrogenase were included to utilise NADH, thereby inhibiting the activity of prostaglandin A13-reductase (Sun and Armour, 1974), which is dependent upon this cofactor. This precaution was taken because 15-ketoPGEl is a substrate for the reductase (Angg~rd, 1971). The crude enzyme preparation was incubated at 37°C and the reaction started by adding 1 mg of PGE~ dissolved in 0.2 ml ethanol, giving a substrate concentration of 0.54 mM. Aliquots of the reaction mixture (0.9 ml) were taken immediately (zero time samples) and at appropriate intervals for up to 60 min thereafter. These were added to 12.5 ml of ice-cold ethanol to stop the reaction. 15ketoPGE~ was extracted b y the method of ~,ngg~rd et al. (1971), and assayed by light absorption at 500 nm after the addition of NaOH (final concentration 0.077 M). As no authentic 15-ketoPGE~ was available, calculations of the amount of metabolite present were based on the millimolar extinction coefficient of the colour species (30.3) quoted by Sun and Armour (1974). Using this value, and correcting for recovery as described below, the concentrations of 15-ketoPGE~ found in incubates wherein the conversion of substrate had apparently proceeded to completion were in accordance with the concentration of PGE1 present initially. The efficiency of the extraction procedure was estimated by measuring the recovery of
J.M. EGERTON-VERNON, J.R. BEDWANI 3 H-PGEI (cf. -~,ngg~ird et al. 1971). This was found to be 75.1 -+ 1.1% (mean -+ S.E., n = 5), and the results were corrected accordingly. 3. Results
The accumulation of 15-ketoPGEj in enzyme preparations from all organs except the liver (both species) and the pregnant rabbit lung was found to increase linearly with time throughout the period of incubation (30--60 min). With the pregnant rabbit lung, the rate of 15-ketoPGE1 accumulation was very rapid and was linear only for a short time (2.5--7.5 min), after which it declined. Calculations of the a m o u n t of metabolite produced showed that this decline could be attributed to depletion of the substrate. With the liver, it was difficult to tell whether the metabolite was produced at a uniform rate, because the amounts detected after incubations of up to 60 min were too small for us to measure accurately. The specific activity of PGDH, calculated as the initial rate of 15-ketoPGE1 formation (pmole/mg protein/min), in rabbit kidney, spleen and lung supernatants is shown in fig. la. With the kidney and spleen, there was no substantial difference between PGDH activity in preparations from pregnant as compared with non-pregnant animals. Although we were unable to measure liver PGDH activity accurately, there was no indication that this changed markedly during pregnancy either. However, in agreement with previous results (Sun and Armour, 1974), lung PGDH activity showed a striking increase in the pregnant rabbits. The results obtained with kidney, spleen and lung from pregnant and non-pregnant (dioestrus) rats are shown in fig. lb. PGDH activity in these organs was considerably lower than in the corresponding organs from the rabbit. Again, we were unable to measure liver PGDH activity accurately, b u t there was no indication that this changed substantially during pregnancy. With the kidney, there was no large difference between PGDH activity in preparations from pregnant and non-pregnant rats, although
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Fig. I. N A D +-dependent prostaglandin 15-hydroxydehydrogenase activity in organs f r o m non-pregnant (open columns and open circles) and pregnant (hatched columns and filled circles) rabbits and rats. Non-pregnant rats were used when in dioestrus; pregnant animals were used on day 26 (rabbits) or day 19 (rats) of gestation. Each column represents the mean result from 3 experiments, each experiment being carried out with organs from individual rabbits or groups of 2--3 rats. The circles indicate the range of results.
there was some evidence of a slight reduction in renal PGDH activity in the pregnant animals. PGDH activity in the spleen was essentially the same in pregnant and non-pregnant groups. In contrast with the result in the rabbit, however, lung PGDH activity was also similar in pregnant and non-pregnant animals•
4. Diseussion
These results suggest that the striking increase in PGDH activity occurring in the rabbit lung during pregnancy does not reflect a gener-
407
al increase in the activity of this enzyme throughout the b o d y , as there was no substantial change in PGDH activity in the spleen or kidney. There was no evidence of any marked increase in liver PGDH activity during pregnancy either; the yield of 15-ketoPGEj from this organ was extremely low in both pregnant and non-pregnant rabbits. At present, the stimulus for the increase in PGDH activity in the rabbit lung during pregnancy is unknown. If this change is caused by a circulating hormone or 'messenger', it is surprising that it does not also occur in other organs. It is possible that the enzyme differs slightly between different organs, at least as far as susceptibility to induction or activation is concerned. Another possibility is that the 'messenger' is removed from the blood during passage through the lung. Plasma prostaglandin F2 a levels in the rabbit show a significant increase between days 21--30 of pregnancy (Challis et al., 1973), and it has been suggested that the stimulus for the enhanced lung activation of prostaglandins is an increased concentration of these c o m p o u n d s in the venous blood (Bedwani and Marley, 1975). The present results support this hypothesis, since the lung efficiently metabolises circulating prostaglandins of the E and F series (Ferreira and Vane, 1967), and thus prevents substantial amounts from reaching the organs• Therefore, if it is these prostaglandins which mediate an increase in PGDH activity, it is appropriate that this change should occur only in the lung. We observed an interesting and unexpected species difference, as there was no increase in lung PGDH activity in the near-term pregnant rat (the normal period of gestation in this species being 21--22 days). In the rabbit, the ability of the lung to inactivate prostaglandin E2 is at its highest during the latter stages of pregnancy (Bedwani and Marley, 1974, 1975), which lasts for 32 days. Sun and Armour (1974) showed that the enhanced metabolism of prostaglandins by the pregnant rabbit lung is due mainly to an increase in PGDH activity. It was suggested that this might constitute a mechanism to afford extra protection against
408
circulating prostaglandins towards the end of gestation. From the present work, it would appear that no such mechanism operates in the rat.
Acknowledgement The authors thank Dr. J.E. Pike of the Upjohn Company, Kalamazoo, Michigan, for a gift of PGEI.
References ~ngg~lrd, E., 1971, Studies on the analysis and metabolism of the prostaglandins, Ann. N.Y. Acad. Sci. 180, 200. ~.ngg~rd, E., C. Larsson and B. Samuelsson, 1971, The distribution of 15-hydroxyprostaglandin de-
J.M. EGERTON-VERNON, J.R. BEDWANI hydrogenase and prostaglandin-A13-reductase in tissues of the swine, Acta Physiol. 81,396. Bedwani, J.R. and P.B. Marley, 1974, Increased inactivation of prostaglandin E 2 by the rabbit lung during pregnancy, Brit. J. Pharmacol. 50,459P. Bedwani, J.R. and P.B. Marley, 1975, Enhanced inactivation of prostaglandin E2 by the rabbit lung during pregnancy or progesterone treatment, Brit. J. Pharmacol. 53, 547. Challis, J.R.G., I.J. Davies and K.J. Ryan, 1973, The relationship between progesterone and prostaglandin F concentrations in the plasma of pregnant rabbits, Prostaglandins 4, 509. Ferreira, S.H. and J.R. Vane, 1967, Prostaglandins: their disappearance from and release into the circulation, Nature (London) 216, 868. Goa, J., 1953, A micro biuret method for protein determination, Scand. J. Clin. Lab. Invest. 5, 218. Sun, F.F. and S.B. Armour, 1974, Prostaglandin 15hydroxydehydrogenase and A13-reductase levels in the lungs of maternal, fetal and neonatal rabbits, Prostaglandins 7, 327.
Short communication PROSTAGLANDIN 15-HYDROXYDEHYDROGENASE ACTIVITY DURING PREGNANCY IN RABBITS AND RATS JULIAN M. EGERTON-VERNON* and JOHN R. BEDWANI**
Department of Pharmacology, University of Cambridg e, Medical School, Hills Road, Cambridge CB2 2QD, U.K. Received 26 June 1975, accepted 24 July 1975
J.M. EGERTON-VERNON and J.R. BEDWANI, Prostaglandin 15-hydroxydehydrogenase activity during pregnancy in rabbits and rats, European J. Pharmacol. 33 (1975) 405--408. Prostaglandin 15-hydroxydehydrogenase (PGDH) activity in the rabbit lung increased strikingly during pregnancy. This change did not occur in the kidney, or spleen. In the corresponding organs from the rat, PGDH activity was essentially the same in pregnant and non-pregnant animals. These results are discussed in relation to the hypothesis that the increased PGDH activity in the pregnant rabbit lung is secondary to an elevated concentration of prostaglandins in the venous blood. Prostaglandin El
Prostaglandin metabolism
1. Introduction The ability of the rabbit lung to inactivate prostaglandin E2 increases markedly during pregnancy (Bedwani and Marley, 1974). This change is associated with a large increase in NAD +- dependent prostaglandin 15-hydroxydehydrogenase (PGDH) activity, and a much smaller increase in prostaglandin A13-reductase activity (Sun and Armour, 1974 ,)The factor responsible for this is not known. Bedwani and Marley (1975) found that chronic treatment of non-pregnant rabbits with progesterone was followed by an increase in lung prostaglandin-metabolizing activity, but thought it unlikely that there is a direct relationship between plasma progesterone levels and prostaglandin metabolism in this organ. * Present address: Middlesex Hospital Medical School, Mortimer Street, London W1, U.K. ** Present address: Department" of Physiology, University College, P.O. Box 78, Cardiff CF1 l X L U.K. To whom requests for reprints should be sent.
Pregnancy
The present experiments were carried out to see whether an increased PGDH activity during pregnancy is confined to the lung, or whether it also occurs in other organs. Accordingly, we investigated the activity of this enzyme in lung, kidney, spleen and liver from pregnant and non-pregnant rabbits. For comparative purposes, similar experiments were also performed in the rat.
2. Materials and methods
Animals used were New Zealand White rabbits and Wistar rats. With the latter species, organs from 2 to 3 animals were pooled for each experiment. Non-pregnant rats were used when in dioestrus. Pregnant animals were sacrificed on day 19 (rats) or day 26 (rabbits) of gestation. PGDH activity was assayed by measuring the formation of 15-ketoprostaglandin E1 on incubation of cytoplasmic fractions from organ homogenates with prostaglandin E1 (PGE1)
406 and NAD *. The procedure was based on the method of .~,ngg~d et al. (1971). Organs were homogenized in 4 volumes of ice-cold buffer of the following composition (mM): K:HPO4 78.3; KH2 PO4 21.7; EDTA 1.0; mercaptoethanol 8.0. A high speed supernatant was prepared from each homogenate by centrifugation at 1400 g for 3 min, then at 100,000 g for I hr (at 4 °C). Aliquots of the supernatant were taken for protein assay (Goa, 1953), and to 5 ml of the remainder were added NAD ÷, sodium pyruvate and lactate dehydrogenase to give final concentrations of 10 mM, 50 mM and 50 pg/ml respectively. The sodium pyruvate and lactate dehydrogenase were included to utilise NADH, thereby inhibiting the activity of prostaglandin A13-reductase (Sun and Armour, 1974), which is dependent upon this cofactor. This precaution was taken because 15-ketoPGEl is a substrate for the reductase (Angg~rd, 1971). The crude enzyme preparation was incubated at 37°C and the reaction started by adding 1 mg of PGE~ dissolved in 0.2 ml ethanol, giving a substrate concentration of 0.54 mM. Aliquots of the reaction mixture (0.9 ml) were taken immediately (zero time samples) and at appropriate intervals for up to 60 min thereafter. These were added to 12.5 ml of ice-cold ethanol to stop the reaction. 15ketoPGE~ was extracted b y the method of ~,ngg~rd et al. (1971), and assayed by light absorption at 500 nm after the addition of NaOH (final concentration 0.077 M). As no authentic 15-ketoPGE~ was available, calculations of the amount of metabolite present were based on the millimolar extinction coefficient of the colour species (30.3) quoted by Sun and Armour (1974). Using this value, and correcting for recovery as described below, the concentrations of 15-ketoPGE~ found in incubates wherein the conversion of substrate had apparently proceeded to completion were in accordance with the concentration of PGE1 present initially. The efficiency of the extraction procedure was estimated by measuring the recovery of
J.M. EGERTON-VERNON, J.R. BEDWANI 3 H-PGEI (cf. -~,ngg~ird et al. 1971). This was found to be 75.1 -+ 1.1% (mean -+ S.E., n = 5), and the results were corrected accordingly. 3. Results
The accumulation of 15-ketoPGEj in enzyme preparations from all organs except the liver (both species) and the pregnant rabbit lung was found to increase linearly with time throughout the period of incubation (30--60 min). With the pregnant rabbit lung, the rate of 15-ketoPGE1 accumulation was very rapid and was linear only for a short time (2.5--7.5 min), after which it declined. Calculations of the a m o u n t of metabolite produced showed that this decline could be attributed to depletion of the substrate. With the liver, it was difficult to tell whether the metabolite was produced at a uniform rate, because the amounts detected after incubations of up to 60 min were too small for us to measure accurately. The specific activity of PGDH, calculated as the initial rate of 15-ketoPGE1 formation (pmole/mg protein/min), in rabbit kidney, spleen and lung supernatants is shown in fig. la. With the kidney and spleen, there was no substantial difference between PGDH activity in preparations from pregnant as compared with non-pregnant animals. Although we were unable to measure liver PGDH activity accurately, there was no indication that this changed markedly during pregnancy either. However, in agreement with previous results (Sun and Armour, 1974), lung PGDH activity showed a striking increase in the pregnant rabbits. The results obtained with kidney, spleen and lung from pregnant and non-pregnant (dioestrus) rats are shown in fig. lb. PGDH activity in these organs was considerably lower than in the corresponding organs from the rabbit. Again, we were unable to measure liver PGDH activity accurately, b u t there was no indication that this changed substantially during pregnancy. With the kidney, there was no large difference between PGDH activity in preparations from pregnant and non-pregnant rats, although
PROSTAGLANDIN
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Fig. I. N A D +-dependent prostaglandin 15-hydroxydehydrogenase activity in organs f r o m non-pregnant (open columns and open circles) and pregnant (hatched columns and filled circles) rabbits and rats. Non-pregnant rats were used when in dioestrus; pregnant animals were used on day 26 (rabbits) or day 19 (rats) of gestation. Each column represents the mean result from 3 experiments, each experiment being carried out with organs from individual rabbits or groups of 2--3 rats. The circles indicate the range of results.
there was some evidence of a slight reduction in renal PGDH activity in the pregnant animals. PGDH activity in the spleen was essentially the same in pregnant and non-pregnant groups. In contrast with the result in the rabbit, however, lung PGDH activity was also similar in pregnant and non-pregnant animals•
4. Diseussion
These results suggest that the striking increase in PGDH activity occurring in the rabbit lung during pregnancy does not reflect a gener-
407
al increase in the activity of this enzyme throughout the b o d y , as there was no substantial change in PGDH activity in the spleen or kidney. There was no evidence of any marked increase in liver PGDH activity during pregnancy either; the yield of 15-ketoPGEj from this organ was extremely low in both pregnant and non-pregnant rabbits. At present, the stimulus for the increase in PGDH activity in the rabbit lung during pregnancy is unknown. If this change is caused by a circulating hormone or 'messenger', it is surprising that it does not also occur in other organs. It is possible that the enzyme differs slightly between different organs, at least as far as susceptibility to induction or activation is concerned. Another possibility is that the 'messenger' is removed from the blood during passage through the lung. Plasma prostaglandin F2 a levels in the rabbit show a significant increase between days 21--30 of pregnancy (Challis et al., 1973), and it has been suggested that the stimulus for the enhanced lung activation of prostaglandins is an increased concentration of these c o m p o u n d s in the venous blood (Bedwani and Marley, 1975). The present results support this hypothesis, since the lung efficiently metabolises circulating prostaglandins of the E and F series (Ferreira and Vane, 1967), and thus prevents substantial amounts from reaching the organs• Therefore, if it is these prostaglandins which mediate an increase in PGDH activity, it is appropriate that this change should occur only in the lung. We observed an interesting and unexpected species difference, as there was no increase in lung PGDH activity in the near-term pregnant rat (the normal period of gestation in this species being 21--22 days). In the rabbit, the ability of the lung to inactivate prostaglandin E2 is at its highest during the latter stages of pregnancy (Bedwani and Marley, 1974, 1975), which lasts for 32 days. Sun and Armour (1974) showed that the enhanced metabolism of prostaglandins by the pregnant rabbit lung is due mainly to an increase in PGDH activity. It was suggested that this might constitute a mechanism to afford extra protection against
408
circulating prostaglandins towards the end of gestation. From the present work, it would appear that no such mechanism operates in the rat.
Acknowledgement The authors thank Dr. J.E. Pike of the Upjohn Company, Kalamazoo, Michigan, for a gift of PGEI.
References ~ngg~lrd, E., 1971, Studies on the analysis and metabolism of the prostaglandins, Ann. N.Y. Acad. Sci. 180, 200. ~.ngg~rd, E., C. Larsson and B. Samuelsson, 1971, The distribution of 15-hydroxyprostaglandin de-
J.M. EGERTON-VERNON, J.R. BEDWANI hydrogenase and prostaglandin-A13-reductase in tissues of the swine, Acta Physiol. 81,396. Bedwani, J.R. and P.B. Marley, 1974, Increased inactivation of prostaglandin E 2 by the rabbit lung during pregnancy, Brit. J. Pharmacol. 50,459P. Bedwani, J.R. and P.B. Marley, 1975, Enhanced inactivation of prostaglandin E2 by the rabbit lung during pregnancy or progesterone treatment, Brit. J. Pharmacol. 53, 547. Challis, J.R.G., I.J. Davies and K.J. Ryan, 1973, The relationship between progesterone and prostaglandin F concentrations in the plasma of pregnant rabbits, Prostaglandins 4, 509. Ferreira, S.H. and J.R. Vane, 1967, Prostaglandins: their disappearance from and release into the circulation, Nature (London) 216, 868. Goa, J., 1953, A micro biuret method for protein determination, Scand. J. Clin. Lab. Invest. 5, 218. Sun, F.F. and S.B. Armour, 1974, Prostaglandin 15hydroxydehydrogenase and A13-reductase levels in the lungs of maternal, fetal and neonatal rabbits, Prostaglandins 7, 327.