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Comparison of the pulmonary vascular response to prostaglandin A1, prostaglandin F2α and angiotensin II; Metabolism of PGA1 in lung
Pergamon Press
Life Sciences Vol . 19, pp . 1653-1662, 1976 . Printed in the U.S .A .
COMPARISON OF THE PULMONARY VASCULAR RESPONSE TO PROSTAGLANDIN A1 , PROSTAGLANDIN F 2a AND ANGIOTENSIN II ;
METABOLISM OF PGAl IN LUNG 1
Renneth B . Gross2 and C . N . Gillis Departments of Anesthesiology and Pharmacology Yale University School of Medicine 333 Cedar Street New Haven, Connecticut 06510 (Received in final form August 26, 1976) Summary A rabbit lung preparation, perfused in vitro , was used to examine pulmonary metabolism of prostaglandin Al (PGA1) and to compare the vasoconstrictor actions of PGAl , prostaglandin F2 a (PGF2a) and angiotensin II . PGF2a caused significantly more, and angiotensin II significantly leas, vasoconstriction than did an equimolar concentration of PGAl . Of three likely PGAl metabolites only 15-keto-PGAl had any significant vasoconstrictor action . Furoaemide and aminophylline (10-3 M) reduced PGAl, PGF2 a Diphloretin phosphate or angiotensin II-induced vasoconstriction . potentiated the vascular effect of angiotensin II . Furoaemide (10 3 H) and DPP (9 .5 x 10 -6 M) significantly reduced pulmonary metabolism of PGAl while aminophylline (10-3 M) had no effect on this process . Perfusion of the lungs with a hypoxic medium had no effect on PGAl metabolism . Prostaglandin A1 (PGAl), at concentrations greater than 2 uM, causes pulmonary vasoconstriction which is antagonized by diphloretin phosphate (DPP), an inhibitor of prostaglandin metabolism (1,2) . Such an inhibitor would be expected to preserve prostaglandin and thus potentiate or prolong its vasoconstrictor action . Possibly, therefore, DPP also directly inhibits PGAlinduced vasoconstriction by a spasmolytic action on vascular muscle . Alternatively, pulmonary vasoconstriction may be caused by a metabolite of PGAl rather than by the prostaglandin itself ; in this event, DPP could be viewed as inhibiting the vasoconstrictor response to PGA1 by preventing its transformation to a bioactive metabolite(s) . We have now evaluated this possibility by determining the vasoconstrictor potency of possible PGAl metabolites, and by comparing the effects of other inhibitors of PG metabolism, namely furosemide (3) and aminophylline (4), on vasoconstrictor response to PGAl and also to PGF2a and angiotensin II . 1 This work was supported by PHS Grant HL 13315 . Training Grant HL 05942 . 2
Present Address :
RBG was supported by PHS
Department of Biological Sciences, Wellesley College,
Wellesley, MA 02181 . 1653
1654
Proataglandins and Perfused Rabbit Luag
Vol . 19, No . 11
Bito (5,6) suggested that prostaglandins are transported into cells _via a saturable energy-dependent process . Accordingly, the effect of hypoxia on pulmonary degradation of PGAl was examined to determine whether oxygen lack interferes with prostaglandin transport from the pulmonary vascular space . When rabbit lungs are perfused with PGAl about 35-45X of the total radioactivity in lung effluent is insoluble in ethyl acetate, the solvent normally used to extract prostaglandins and metabolite (1) . This may be due to forma tion of a water soluble adduct of reduced glutathione with PGAl and its metabolite s) (7,8) . It was therefore of interest to determine whether the presumed formation of adduct was altered by any of the drugs or other treatments of lung used in this study . Materials and Methods Perfused rabbit lung preparation . The technique used for simultaneous perfusion of right and left lunge _in vitro has been described in detail (2,9) . In brief, lungs were perfused simultaneously but independently at a flow rate of 10 ml/min for each lung, through the first branches of the pulmonary artery with a Rrebs-bicarbonate buffer (see below) at 37 °C . Lungs were inflated through the trachea with 20-22 cc of room air . Arterial perfusion pressure was monitored continuously by means of Statham P 23 transducers and a Grass polygraph. Since flow was constant (Holter pump, Model #P 1110), changes in arterial perfusion pressure reflected altered pulmonary vascular resistance . In all experiments lunge were perfused for 10 minutes (equilibration period) with Rrebs medium before starting perfusion with drugs and/or prostaglandina . During this period, perfusion pressure stabilized between 3 and 8 mm Hg . Whenever the effect of drugs on prostaglandin metabolism was examined, the drug under study was perfused through one lung (the experimental lung) for at least 5 minutes prior to its co-perfusion with prostaglandin . The contralateral (i .e ., control) lung was simultaneously perfused with Krebs medium during the entire period . Hypoxia. In experiments involving perfusion with "low oxygen" Krebs medium, distilled water was boiled for at least 40 minutes and then aerated continuously with 95X N2 - 5X C02 during both addition of the required salts and lung perfusion. The p02 of Krebs prepared in this manner was between 20 and 40 mm Hg both before and during the experiments; the comparable value was 400-500 mm Hg in Rrebs medium aerated with 95X 02 - 5X C02 . In all experiments involving hypoxia, experimental lungs were perfused with low oxygen Krebs medium for at least 20 minutes before perfusion of the PGAl dissolved in the same medium ; simultaneously the control lung was perfused with oxygenated Krebs . Experimental Design . a)
PG metabolism experiments :
Since the lung preparation permits simultaneous but independent perfusion and collection of effluent from each lung, one of each pair of lungs was used as a control (e .g ., perfused with PGA 1) while the contralateral served as experimental lung (e .g ., perfused with PGAl and a metabolic inhibitor) . The difference between the two lungs then reflected the effect of the inhibitor. For example, when four pairs of lungs were used to evaluate the effect of a drug on prostaglandin metabolism, values for percent metabolism in two left~and two right lunge were used to determine mean control metabolism, while data from
Vol . 19, No . 11
Proataglandina and Perfused Rabbit Lsmg
1655
corresponding lungs perfuaed with prostaglandin and the drug yielded data for mean percent metabolism in the presence or the inhibitor, b)
Pulmonary vasocons trictor responses :
In moat experiments where pulmonary vasoconstrictor responses to prostaglandin or angiotenain II were measured in the absence (control) and presence (experimental) of a drug, both responses were elicited in the same Inhibitors were always lung to avoid inter-lung differences in sensitivity . perfuaed for at least 5 minutes before co-perfusing them with prostaglandin or angiotenain II . Measurement of pros taglandin and metabolites in lung effluent . Methods for extraction and measurement of prostaglandin and metabolites Lungs were perfuaed with in lung effluent have been described in detail (2) . radioactive prostaglandin after which metabolite s) was extracted from the The ethyl acetate was evaporated to acidified effluent into ethyl acetate. dryness and the residue redissolved in a small amount of methanol . Prostaglandin and metabolite s) were separated by thin layer chromatography (Silica gel) and the location of each determined by means of marker samples. Radioactivity corresponding to each peak was measured by liquid scintillation spectrometry . Percent metabolism was calculated as that radioactivity corresponding to metabolite divided by the sum of total radioactivity representing metabolite and unchanged prostaglandin . The difference between radioactivity in lung effluent before and after the extraction with ethyl acetate reflected that isotope associated with compound (s) insoluble in the latter solvent . Data are reported as the mean _+ standard error of the mean . significance was determined using Student's "t" test (10) .
Statistical
Perfusion medium and drugs. Rrebs bicarbonate medium had the following composition (millimolar concentration) : NaCl, 118 .07 ; RC1, 4 .75 ; CaC12, 2 .54 ; KH2P04, 1 .19 ; MgS04, 1 .10 ; NaHCOg, 25 .0 ; glucose, 11 .1 . Radioactive [5,6-3H]prostaglandin A1 (specific activity > 60 C/mmol) and aminophylline were purchased from New England Nuclear Corp ., Boston, Mass . and Schwarz/Mann, Orangeburg, N .Y ., respectively . Unlabeled prostaglandin and metabolites were obtained from the Upjohn Co ., Kalamazoo, Mich . ; furosemide from Hoechst-Rousael Pharmaceuticals, Inc., Somerville, N.J ., and diphloretin phosphate from AB Leo, Sweden . Results In table 1 the pulmonary vascular response to PGAl is compared with that produced by PGF2 o , angiotenain II and 15-keto-PGA1 . Prostaglandin F2 a (3 uM) had a significantly greater effect (P < 0.05) on vascular resistance than did an equimolar concentration of PGAl in the same lung ; the action of angiotenain II (3 uM) was less than that of PGA1 (P < 0 .005) . Although perfuaed at twice the PGAl concentration, the vasoconstrictor effect of 15-keto-PGA1 (6 .0 uM) was significantly less than that of the former (P < 0 .05) . Neither 13,14-dihydro15-keto-PGA l nor 13,14-dihydro-PGAl had any observable effect on vascular resistance .
1656
Vol . 19, No . 11
Prostaglandins and Perfused Rabbit Lung TABLE 1
Vasoconstrictor Effects of Prostaglandin F2aa , Angiotensin II a a and 15-keto-PGAi, Compared with Prostaglandin Al Lunge were perfuaed with Kreba medium for 10-15 minutes (equilibration period) and then perfuaed alternately with PGAi and one of the other substances, each for 2 .5-3 minutes . A period of 10 minutes was allowed between each drug perfusion for vascular resistance to return to control levels . Increase in pulmonary mean arterial perfusion pressure (mm Hg) PGAi PGF2a
a b
18 .3 + 3 .2c
(n=8)
14 .1 + 2 .8c
Angiotensin II
4 .9 + 0 .7d
(n=8)
19 .8 + 3 .7d
15-keto-PGAi
5 .3 + 0 .97e
(n=4)
10 .3 + 1 .7e
Perfused at 3 x 10 Perfused at 6 x 10
6 6
uM yM
c P < 0.05 (paired t-test) d P < 0.005 (paired t-test) e P < 0 .05 (paired tpteat)
(10
3 3 Table 2 indicates that furoaemide M), aminophylline M) and -6 diphloretin phosphate (9 .5 x 10 M) significantly decreased the vascular response to PGAi and PGF2a , Pulmonary metabolism of PGAi was also significantly reduced by furoaemide and DPP, but was unaffected by aminophylline. There was no obvious correlation between inhibition of metabolism and inhibition of vasoconstriction, Table 2 also shows that furoaemide and aminophylline significantly reduced the vasoconstrictor response to angiotenain II (P < 0 .005 and P < 0 .05, respectively) . Surprisingly however, DPP potentiated angiotenain II-induced vasoconstriction (P < 0 .005),
(10
From table 3, it can be seen that DPP significantly (P < 0 .02) increased the amount of radioactivity, in effluent of lungs perfuaed with ~-pGAl, which was soluble in ethyl acetate . Hypothermia was more effective in this regard and resulted in quantitative extraction of radioactivity into ethyl acetate (table 3) . Percent metabolism of PGAi in both "hypoxic" and contralateral control lunge was 65 _+ 1X (n=3) . Figure 1 shows the rates of appearance in the effluent (collected during 1-minute intervals) of unchanged PGAi, metabolized PGAi and also the ethyl acetate-insoluble radioactivity in the effluent during 8 minutes of perfusion with PGAi (0 .3 uM) and a subsequent 6-minute washout period . It is clear (figure . la) that only a small portion of the PGAi perfuaed through the
Vol . 19, No . 11
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Prostaglandine and Perfused Rabbit Lung
Vol . 19, No . 11
TABLE 3 Percent of Radioactivity,
in Effluent of Lungs Perfused with PGAl ,
which was Extracted from Kreba into Ethyl Acetate s Experimental Treatment
Control
Experimental
Aminophyllineb (10-3 M; n=4)
73 + 5 -
76 + 5
-3 Furosemidé (10 M; n=4)
64 + 4
64 + 4
Hypothermiac (n~3)
66 + 4
PGElc (3 x 10 Hypoxiac (n~3) a b
6
; n-3)
*
99 + 0 .3
67 + 3
69 + 6
55 + 2
52 + 5
Lung effluent was washed twice with equal volumes of ethyl acetate. 6 PGAl perfused at 3 x 10 M concentration.
c PGAl perfused at 3 x 10 7 M concentration. Control and experimental values significantly different (P < 0 .02) .
lung is recovered unchanged . Furthermore, similar amounts of metabolite and "non-extractable" tritium are recovered in each fraction . To allow comparison of relative rates of initial appearance and efflux of each form of radioactivity in the effluent after the end of PGAl perfusion, the data of figure Ta are normalized . For this purpose, the maximum tritium corresponding to each substance recovered in all fractions was arbitrarily assigned the value of 100 and radioactivity of other fractions was expressed as a percentage of this maximum. Rates of appearance and washout of PGAl in its "extractable" metabolite coincide closely (figure lb) . However, the nonextractable tritium is somewhat more slowly lost from lung . Discussion Angiotenain II is generally accepted to be one of "the moat potent of known vasoconstrictor agents" (11) . However, in perfused rabbit lung PGAl and The relative PGF2 a were several fold more potent than angiotensin II . potencies of PGF2 and angiotensin II we observed are in accord with previous reports (12), alt~ough the pulmonary vasoconstrictor effect of PGAl (table 1) contrasts with the potent vasodilator action of PGA1 in the dog lung in situ (12 ). The fact that the possible prostaglandin Al metabolites are much less potent than PGAl indicates that PGAl-induced pulmonary vasoconstriction is not However, lung may take up and due to formation of these substances in lung . metabolize prostaglandin to products which are then released at sites very close
Prostaglandins and Perfused Rabbit Lung
Vol. 19, No . 11
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1659
20 18 12 8 4 O
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FIG. 1 Lungs were perfuaed for 8 minutes with 3H-PGA (0 .3 yM) and then for 6 minutes with Kreba medium . (A} shows the appearance of unchanged PGA1 ("), metabolized PGA (0) and tritium which was insoluble in ethyl acetate ~~) in fractions of effluent collected for one minute intervals . Data are expressed as DPM, corresponding to each substance . In (B) the recovery of each substance is expressed in arbitrary units . The highest DPM recovered for each substance is assigned the value of 100 . Radioactivity in other fractions is expressed as a percentage of this maximum. to a prostaglandin receptor . Thus, the concentration of metabolite in the microenvironment at the site of vasoconstrictor action would be greater than anticipated . Furthermore, the concentration of 15-keto-PGAl perfuaed (6 uM) may be somewhat lower than that produced at strategic sites in the vasculature
1660
Prostaglandins and Perfuaed Rabbit Lung
when lungs are perfused with PGAl
Vol . 19, No . 11
(3 uM) .
Although there is no information concerning possible prostaglandin receptors in the lung, there is some evidence for a specific high affinity prostaglandin receptor in other tissues (13) . The possibility that DPP inhibits PG-induced vasoconstriction in the lung by blocking a "receptor", suggests a reasonably specific action, since the former poteatiated rather than inhibited angiotensin II-induced vasoconstriction . The cause of this potentiation is unclear . Furosemide and aminophylline inhibit purified prostaglandin dehydrogeaase, the enzyme responsible for the initial step in prostaglandin degradation (3,4) . In our studies, furoaemide inhibited both the metabolism and pulmonary vaso constriction caused by the prostaglandins . Inhibition of proataglandiniaduced vasoconstriction was non-specific since furoaemide had a similar action on the constrictor effects of angiotensin II . Aminophylline failed to inhibit PG metabolism in the intact lung . Possibly the difference between our results and the report of Marrazzi and Matschinaky (4) reflects the fact that, in the intact lung used in our work, aminophylline could not reach the enzyme where it presumably acts . Inhibition of prostaglandin or angiotensin II-induced vasoconstriction by aminophylline more likely is related to the generalized smooth muscle relaxant properties of aminophylline (14) . Both DPP and hypothermic perfusion significantly reduced the amount of radioactivity in lung effluent which was insoluble in ethyl acetate (table 3) . Possibly, DPP reacts ear se with the sulfhydryl group of glutathione, thus inhibiting formation of the PGAl adduct . Alternatively DPP may inhibit glutathione reductase, the enzyme which transforms glutathione from the oxidized to its reactive reduced state . Hypothermia causes a reversible transition in the physical state of lipids within the cellular membranes from liquid to solid state (15), which likely affects membrane permeability and may prevent PGAl from reaching the cellular(?) site of adduct formation . In experiments where lung effluent was fractionated (figure 1), the rate of appearance of extractable PGAl , unchanged PGA l and metabolized PGAl are similar . During washout however, the non-extractable radioactivity lags behind the two other forms . This apparently greater volume of distribution for the "non-extractable PGAl" may be due to a slower exit of the presumed PGAl -glutathione adduct from cells than that of PGAl or its metabolite . Our data indicate the need for considerable caution in examination of PGAl in blood . It is well known that there are relatively large amounts of reduced glutathione circulating in blood (e .g ., 267-319 mg/liter in man, 260-400 mg/liter in rabbit) (16) which could form the adduct with circulating PGAl . Should this occur, the usual methods for prostaglandin extraction from blood, involving use of organic solvents, would not extract the relatively polar glutathione adduct of PGAl . Accordingly, the presence of PGAl in the blood would be underestimated if detected at all . In addition the ability of various vascular beds to remove and/or metabolize PGAl in circulation may depends on whether or not the PGA1 is in its free form . These possibilities suggest that new techniques are needed for analysis of PGAl and its metabolites in blood and other tissues : these should not relay on the relatively non-polar properties of the free prostaglandins . Acknowle dgements The authors express their appreciation for gifts of drugs from : Dr . John Pike, the Upjohn Company ; Dr . Bertil HSgberg, AB Leo ; Dr . Charles Brownley, Ciba-Geigy ; and Dr . Val Wagner, Hoechst-Roussel Pharmaceuticals, Inc .
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We also wish to thank Ms . Margaret Erdmann for her excellent technical assistance . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
R .B . GROSS and C .N . GILLIS, Biochem . Pharmacol . _24 1441-1444 (1975) . K .B . GROSS and C .N . GILLIS, J . Pharmacol . exp . Ther ., in press (1976) . J .R . PAULSRUD and O .N . MILLER, Fed . Proc . 33 590 (1974) . M .A . MARRAZZI and F .M . MATSCHINSKY, Prostaglandins _1 373-388 (1972) . L .Z . BITO, J . Phyaiol . 221 371-387 (1972) . L .Z . BITO, Nature _256 134-136 (1975) . L .M . CAGEN, J .J . PISANO and H .M . FAZES, Fed . Proc . _34 790 (1975a) . L .M . CAGEN, J .J . PISANO, J .N . KETLEY, W .H . HABIG and W .B . JAKOBY, Biochim . Biophys . Acts _398 205-208 (1975b) . C .N . GILLIS and Y . IWASAWA, J . Appl . Phyaiol . _33 404-408 (1972) . A .B . HILL, Principles of Medical Statistics , The Lancet Limited, London (1966) . B . FOLKOW and E . NEIL, Circulation , Oxford University Press, New York (1971) . P .J . KADOWITZ and A .L . HYMAN, Proc . Soc . exp . biol . Med . _149 282-286 (1975) . F .A . KUEHL and J .L . HUMES, Proc . Nat . Acad . Sci . _69 480-484 (1972) . C .W . QUIMBY, M .A . DOMINGO and C .F . SCHMIDT, J . Pharmacol . exp . Ther . _122 396 (1958) . C .F . FOR, MTP International Review of Science . Biochemistry of cell walls and membranes, University Park Press, Baltimore (1975) .
Life Sciences Vol . 19, pp . 1653-1662, 1976 . Printed in the U.S .A .
COMPARISON OF THE PULMONARY VASCULAR RESPONSE TO PROSTAGLANDIN A1 , PROSTAGLANDIN F 2a AND ANGIOTENSIN II ;
METABOLISM OF PGAl IN LUNG 1
Renneth B . Gross2 and C . N . Gillis Departments of Anesthesiology and Pharmacology Yale University School of Medicine 333 Cedar Street New Haven, Connecticut 06510 (Received in final form August 26, 1976) Summary A rabbit lung preparation, perfused in vitro , was used to examine pulmonary metabolism of prostaglandin Al (PGA1) and to compare the vasoconstrictor actions of PGAl , prostaglandin F2 a (PGF2a) and angiotensin II . PGF2a caused significantly more, and angiotensin II significantly leas, vasoconstriction than did an equimolar concentration of PGAl . Of three likely PGAl metabolites only 15-keto-PGAl had any significant vasoconstrictor action . Furoaemide and aminophylline (10-3 M) reduced PGAl, PGF2 a Diphloretin phosphate or angiotensin II-induced vasoconstriction . potentiated the vascular effect of angiotensin II . Furoaemide (10 3 H) and DPP (9 .5 x 10 -6 M) significantly reduced pulmonary metabolism of PGAl while aminophylline (10-3 M) had no effect on this process . Perfusion of the lungs with a hypoxic medium had no effect on PGAl metabolism . Prostaglandin A1 (PGAl), at concentrations greater than 2 uM, causes pulmonary vasoconstriction which is antagonized by diphloretin phosphate (DPP), an inhibitor of prostaglandin metabolism (1,2) . Such an inhibitor would be expected to preserve prostaglandin and thus potentiate or prolong its vasoconstrictor action . Possibly, therefore, DPP also directly inhibits PGAlinduced vasoconstriction by a spasmolytic action on vascular muscle . Alternatively, pulmonary vasoconstriction may be caused by a metabolite of PGAl rather than by the prostaglandin itself ; in this event, DPP could be viewed as inhibiting the vasoconstrictor response to PGA1 by preventing its transformation to a bioactive metabolite(s) . We have now evaluated this possibility by determining the vasoconstrictor potency of possible PGAl metabolites, and by comparing the effects of other inhibitors of PG metabolism, namely furosemide (3) and aminophylline (4), on vasoconstrictor response to PGAl and also to PGF2a and angiotensin II . 1 This work was supported by PHS Grant HL 13315 . Training Grant HL 05942 . 2
Present Address :
RBG was supported by PHS
Department of Biological Sciences, Wellesley College,
Wellesley, MA 02181 . 1653
1654
Proataglandins and Perfused Rabbit Luag
Vol . 19, No . 11
Bito (5,6) suggested that prostaglandins are transported into cells _via a saturable energy-dependent process . Accordingly, the effect of hypoxia on pulmonary degradation of PGAl was examined to determine whether oxygen lack interferes with prostaglandin transport from the pulmonary vascular space . When rabbit lungs are perfused with PGAl about 35-45X of the total radioactivity in lung effluent is insoluble in ethyl acetate, the solvent normally used to extract prostaglandins and metabolite (1) . This may be due to forma tion of a water soluble adduct of reduced glutathione with PGAl and its metabolite s) (7,8) . It was therefore of interest to determine whether the presumed formation of adduct was altered by any of the drugs or other treatments of lung used in this study . Materials and Methods Perfused rabbit lung preparation . The technique used for simultaneous perfusion of right and left lunge _in vitro has been described in detail (2,9) . In brief, lungs were perfused simultaneously but independently at a flow rate of 10 ml/min for each lung, through the first branches of the pulmonary artery with a Rrebs-bicarbonate buffer (see below) at 37 °C . Lungs were inflated through the trachea with 20-22 cc of room air . Arterial perfusion pressure was monitored continuously by means of Statham P 23 transducers and a Grass polygraph. Since flow was constant (Holter pump, Model #P 1110), changes in arterial perfusion pressure reflected altered pulmonary vascular resistance . In all experiments lunge were perfused for 10 minutes (equilibration period) with Rrebs medium before starting perfusion with drugs and/or prostaglandina . During this period, perfusion pressure stabilized between 3 and 8 mm Hg . Whenever the effect of drugs on prostaglandin metabolism was examined, the drug under study was perfused through one lung (the experimental lung) for at least 5 minutes prior to its co-perfusion with prostaglandin . The contralateral (i .e ., control) lung was simultaneously perfused with Krebs medium during the entire period . Hypoxia. In experiments involving perfusion with "low oxygen" Krebs medium, distilled water was boiled for at least 40 minutes and then aerated continuously with 95X N2 - 5X C02 during both addition of the required salts and lung perfusion. The p02 of Krebs prepared in this manner was between 20 and 40 mm Hg both before and during the experiments; the comparable value was 400-500 mm Hg in Rrebs medium aerated with 95X 02 - 5X C02 . In all experiments involving hypoxia, experimental lungs were perfused with low oxygen Krebs medium for at least 20 minutes before perfusion of the PGAl dissolved in the same medium ; simultaneously the control lung was perfused with oxygenated Krebs . Experimental Design . a)
PG metabolism experiments :
Since the lung preparation permits simultaneous but independent perfusion and collection of effluent from each lung, one of each pair of lungs was used as a control (e .g ., perfused with PGA 1) while the contralateral served as experimental lung (e .g ., perfused with PGAl and a metabolic inhibitor) . The difference between the two lungs then reflected the effect of the inhibitor. For example, when four pairs of lungs were used to evaluate the effect of a drug on prostaglandin metabolism, values for percent metabolism in two left~and two right lunge were used to determine mean control metabolism, while data from
Vol . 19, No . 11
Proataglandina and Perfused Rabbit Lsmg
1655
corresponding lungs perfuaed with prostaglandin and the drug yielded data for mean percent metabolism in the presence or the inhibitor, b)
Pulmonary vasocons trictor responses :
In moat experiments where pulmonary vasoconstrictor responses to prostaglandin or angiotenain II were measured in the absence (control) and presence (experimental) of a drug, both responses were elicited in the same Inhibitors were always lung to avoid inter-lung differences in sensitivity . perfuaed for at least 5 minutes before co-perfusing them with prostaglandin or angiotenain II . Measurement of pros taglandin and metabolites in lung effluent . Methods for extraction and measurement of prostaglandin and metabolites Lungs were perfuaed with in lung effluent have been described in detail (2) . radioactive prostaglandin after which metabolite s) was extracted from the The ethyl acetate was evaporated to acidified effluent into ethyl acetate. dryness and the residue redissolved in a small amount of methanol . Prostaglandin and metabolite s) were separated by thin layer chromatography (Silica gel) and the location of each determined by means of marker samples. Radioactivity corresponding to each peak was measured by liquid scintillation spectrometry . Percent metabolism was calculated as that radioactivity corresponding to metabolite divided by the sum of total radioactivity representing metabolite and unchanged prostaglandin . The difference between radioactivity in lung effluent before and after the extraction with ethyl acetate reflected that isotope associated with compound (s) insoluble in the latter solvent . Data are reported as the mean _+ standard error of the mean . significance was determined using Student's "t" test (10) .
Statistical
Perfusion medium and drugs. Rrebs bicarbonate medium had the following composition (millimolar concentration) : NaCl, 118 .07 ; RC1, 4 .75 ; CaC12, 2 .54 ; KH2P04, 1 .19 ; MgS04, 1 .10 ; NaHCOg, 25 .0 ; glucose, 11 .1 . Radioactive [5,6-3H]prostaglandin A1 (specific activity > 60 C/mmol) and aminophylline were purchased from New England Nuclear Corp ., Boston, Mass . and Schwarz/Mann, Orangeburg, N .Y ., respectively . Unlabeled prostaglandin and metabolites were obtained from the Upjohn Co ., Kalamazoo, Mich . ; furosemide from Hoechst-Rousael Pharmaceuticals, Inc., Somerville, N.J ., and diphloretin phosphate from AB Leo, Sweden . Results In table 1 the pulmonary vascular response to PGAl is compared with that produced by PGF2 o , angiotenain II and 15-keto-PGA1 . Prostaglandin F2 a (3 uM) had a significantly greater effect (P < 0.05) on vascular resistance than did an equimolar concentration of PGAl in the same lung ; the action of angiotenain II (3 uM) was less than that of PGA1 (P < 0 .005) . Although perfuaed at twice the PGAl concentration, the vasoconstrictor effect of 15-keto-PGA1 (6 .0 uM) was significantly less than that of the former (P < 0 .05) . Neither 13,14-dihydro15-keto-PGA l nor 13,14-dihydro-PGAl had any observable effect on vascular resistance .
1656
Vol . 19, No . 11
Prostaglandins and Perfused Rabbit Lung TABLE 1
Vasoconstrictor Effects of Prostaglandin F2aa , Angiotensin II a a and 15-keto-PGAi, Compared with Prostaglandin Al Lunge were perfuaed with Kreba medium for 10-15 minutes (equilibration period) and then perfuaed alternately with PGAi and one of the other substances, each for 2 .5-3 minutes . A period of 10 minutes was allowed between each drug perfusion for vascular resistance to return to control levels . Increase in pulmonary mean arterial perfusion pressure (mm Hg) PGAi PGF2a
a b
18 .3 + 3 .2c
(n=8)
14 .1 + 2 .8c
Angiotensin II
4 .9 + 0 .7d
(n=8)
19 .8 + 3 .7d
15-keto-PGAi
5 .3 + 0 .97e
(n=4)
10 .3 + 1 .7e
Perfused at 3 x 10 Perfused at 6 x 10
6 6
uM yM
c P < 0.05 (paired t-test) d P < 0.005 (paired t-test) e P < 0 .05 (paired tpteat)
(10
3 3 Table 2 indicates that furoaemide M), aminophylline M) and -6 diphloretin phosphate (9 .5 x 10 M) significantly decreased the vascular response to PGAi and PGF2a , Pulmonary metabolism of PGAi was also significantly reduced by furoaemide and DPP, but was unaffected by aminophylline. There was no obvious correlation between inhibition of metabolism and inhibition of vasoconstriction, Table 2 also shows that furoaemide and aminophylline significantly reduced the vasoconstrictor response to angiotenain II (P < 0 .005 and P < 0 .05, respectively) . Surprisingly however, DPP potentiated angiotenain II-induced vasoconstriction (P < 0 .005),
(10
From table 3, it can be seen that DPP significantly (P < 0 .02) increased the amount of radioactivity, in effluent of lungs perfuaed with ~-pGAl, which was soluble in ethyl acetate . Hypothermia was more effective in this regard and resulted in quantitative extraction of radioactivity into ethyl acetate (table 3) . Percent metabolism of PGAi in both "hypoxic" and contralateral control lunge was 65 _+ 1X (n=3) . Figure 1 shows the rates of appearance in the effluent (collected during 1-minute intervals) of unchanged PGAi, metabolized PGAi and also the ethyl acetate-insoluble radioactivity in the effluent during 8 minutes of perfusion with PGAi (0 .3 uM) and a subsequent 6-minute washout period . It is clear (figure . la) that only a small portion of the PGAi perfuaed through the
Vol . 19, No . 11
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1658
Prostaglandine and Perfused Rabbit Lung
Vol . 19, No . 11
TABLE 3 Percent of Radioactivity,
in Effluent of Lungs Perfused with PGAl ,
which was Extracted from Kreba into Ethyl Acetate s Experimental Treatment
Control
Experimental
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73 + 5 -
76 + 5
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64 + 4
64 + 4
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66 + 4
PGElc (3 x 10 Hypoxiac (n~3) a b
6
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*
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67 + 3
69 + 6
55 + 2
52 + 5
Lung effluent was washed twice with equal volumes of ethyl acetate. 6 PGAl perfused at 3 x 10 M concentration.
c PGAl perfused at 3 x 10 7 M concentration. Control and experimental values significantly different (P < 0 .02) .
lung is recovered unchanged . Furthermore, similar amounts of metabolite and "non-extractable" tritium are recovered in each fraction . To allow comparison of relative rates of initial appearance and efflux of each form of radioactivity in the effluent after the end of PGAl perfusion, the data of figure Ta are normalized . For this purpose, the maximum tritium corresponding to each substance recovered in all fractions was arbitrarily assigned the value of 100 and radioactivity of other fractions was expressed as a percentage of this maximum. Rates of appearance and washout of PGAl in its "extractable" metabolite coincide closely (figure lb) . However, the nonextractable tritium is somewhat more slowly lost from lung . Discussion Angiotenain II is generally accepted to be one of "the moat potent of known vasoconstrictor agents" (11) . However, in perfused rabbit lung PGAl and The relative PGF2 a were several fold more potent than angiotensin II . potencies of PGF2 and angiotensin II we observed are in accord with previous reports (12), alt~ough the pulmonary vasoconstrictor effect of PGAl (table 1) contrasts with the potent vasodilator action of PGA1 in the dog lung in situ (12 ). The fact that the possible prostaglandin Al metabolites are much less potent than PGAl indicates that PGAl-induced pulmonary vasoconstriction is not However, lung may take up and due to formation of these substances in lung . metabolize prostaglandin to products which are then released at sites very close
Prostaglandins and Perfused Rabbit Lung
Vol. 19, No . 11
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1659
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FIG. 1 Lungs were perfuaed for 8 minutes with 3H-PGA (0 .3 yM) and then for 6 minutes with Kreba medium . (A} shows the appearance of unchanged PGA1 ("), metabolized PGA (0) and tritium which was insoluble in ethyl acetate ~~) in fractions of effluent collected for one minute intervals . Data are expressed as DPM, corresponding to each substance . In (B) the recovery of each substance is expressed in arbitrary units . The highest DPM recovered for each substance is assigned the value of 100 . Radioactivity in other fractions is expressed as a percentage of this maximum. to a prostaglandin receptor . Thus, the concentration of metabolite in the microenvironment at the site of vasoconstrictor action would be greater than anticipated . Furthermore, the concentration of 15-keto-PGAl perfuaed (6 uM) may be somewhat lower than that produced at strategic sites in the vasculature
1660
Prostaglandins and Perfuaed Rabbit Lung
when lungs are perfused with PGAl
Vol . 19, No . 11
(3 uM) .
Although there is no information concerning possible prostaglandin receptors in the lung, there is some evidence for a specific high affinity prostaglandin receptor in other tissues (13) . The possibility that DPP inhibits PG-induced vasoconstriction in the lung by blocking a "receptor", suggests a reasonably specific action, since the former poteatiated rather than inhibited angiotensin II-induced vasoconstriction . The cause of this potentiation is unclear . Furosemide and aminophylline inhibit purified prostaglandin dehydrogeaase, the enzyme responsible for the initial step in prostaglandin degradation (3,4) . In our studies, furoaemide inhibited both the metabolism and pulmonary vaso constriction caused by the prostaglandins . Inhibition of proataglandiniaduced vasoconstriction was non-specific since furoaemide had a similar action on the constrictor effects of angiotensin II . Aminophylline failed to inhibit PG metabolism in the intact lung . Possibly the difference between our results and the report of Marrazzi and Matschinaky (4) reflects the fact that, in the intact lung used in our work, aminophylline could not reach the enzyme where it presumably acts . Inhibition of prostaglandin or angiotensin II-induced vasoconstriction by aminophylline more likely is related to the generalized smooth muscle relaxant properties of aminophylline (14) . Both DPP and hypothermic perfusion significantly reduced the amount of radioactivity in lung effluent which was insoluble in ethyl acetate (table 3) . Possibly, DPP reacts ear se with the sulfhydryl group of glutathione, thus inhibiting formation of the PGAl adduct . Alternatively DPP may inhibit glutathione reductase, the enzyme which transforms glutathione from the oxidized to its reactive reduced state . Hypothermia causes a reversible transition in the physical state of lipids within the cellular membranes from liquid to solid state (15), which likely affects membrane permeability and may prevent PGAl from reaching the cellular(?) site of adduct formation . In experiments where lung effluent was fractionated (figure 1), the rate of appearance of extractable PGAl , unchanged PGA l and metabolized PGAl are similar . During washout however, the non-extractable radioactivity lags behind the two other forms . This apparently greater volume of distribution for the "non-extractable PGAl" may be due to a slower exit of the presumed PGAl -glutathione adduct from cells than that of PGAl or its metabolite . Our data indicate the need for considerable caution in examination of PGAl in blood . It is well known that there are relatively large amounts of reduced glutathione circulating in blood (e .g ., 267-319 mg/liter in man, 260-400 mg/liter in rabbit) (16) which could form the adduct with circulating PGAl . Should this occur, the usual methods for prostaglandin extraction from blood, involving use of organic solvents, would not extract the relatively polar glutathione adduct of PGAl . Accordingly, the presence of PGAl in the blood would be underestimated if detected at all . In addition the ability of various vascular beds to remove and/or metabolize PGAl in circulation may depends on whether or not the PGA1 is in its free form . These possibilities suggest that new techniques are needed for analysis of PGAl and its metabolites in blood and other tissues : these should not relay on the relatively non-polar properties of the free prostaglandins . Acknowle dgements The authors express their appreciation for gifts of drugs from : Dr . John Pike, the Upjohn Company ; Dr . Bertil HSgberg, AB Leo ; Dr . Charles Brownley, Ciba-Geigy ; and Dr . Val Wagner, Hoechst-Roussel Pharmaceuticals, Inc .
Vol . 19, No . 11
Prostaglandins and Perfused Rabbit Lung
1661
We also wish to thank Ms . Margaret Erdmann for her excellent technical assistance . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
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