PROSTAGLANDINS

PROSTAGLANDIN

REMOVAL AND METABOLISM

BY

ISOLATED PERFUSED RAT LUNG

M. W. Anderson

and T. E. Eling

(With the technical assistance

of J. Guthrie and H. Hawkins)

Pharmacokinetics Section Pharmacology and Environmental Biometry Branches National Institute of Environmental Health Sciences Research Triangle Park, North Carolina 27709

ABSTRACT

We have investigated prostaglandins directional

the mechanism(s)

involved in the removal of

(PG) from the pulmonary circulation

fluxes of PG from the circulation

by the lung.

Uni-

into the lung are measured

in an isolated perfused rat lung preparation.

Evidence is presented

which suggests that a transport system for PG exists in lung tissue. This transport system is responsible circulation

by the lung.

for the removal of some PG from the

PGE, and PGF2a are substrates

whereas PGB,, PGA,, and l5-keto-PGFpa for the intracellular metabolism

are not.

PG dehydrogenase,

system for circulating

for this system,

Since PGA, is a substrate

the selectivity

of the lung's

PG is probably due to the selectivity

of the transport system for PG.

It is shown that the percentage of the

pulmonary arterial concentration

(CA) of PGE, or PGF2, that is metabolized

on passage through the pulmonary circulation increases. detected

decreases

When the lungs were perfused with PGE, (PGF2,), the metabolites

in the venous effluent were 15-keto-PGE,

13,14-dihydro-PGE, metabolites

rapidly as CA

(PGF2,).

(PGF2,)

and

15-k&o_

The time course pattern of the appearance

of

in the venous effluent after the initiation of a constant

CA, and the relative concentrations

of the metabolites

in the venous

effluent, were examined as a function of CA. 4-21-76

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ACKNOWLEDGEMENTS The authors wish to acknowledge the technical assistance of David Jones, the assistance of Mary Jo Hicks for data analysis and the assistance of Mary Jo Hicks and Barbara McInnis for their help in preparation of this manuscript. We wish to thank Dr. John Pike, Upjohn Co., for supplying the prostaglandins. INTRODUCTION I t has been well documented that lung tissue of several species is capable of metabolizing prostaglandin (PG) (1,2). Vane and his co-workers (3,4) and other investigators (5,6,7) have shown that the isolated perfused lung as well as lungs in situ extensively degrade circulating PG. Dog and cat lungs metabolize PGE and PGF but not PGA type PG (5,6), while guinea pig lung degrades PGF, PGE, and PGA type PG (3).

In most species,

two enzymes have been implicated in the enzymatic conversion of PG by lung tissue:

(1) a 15-hydroxy-prostaglandin dehydrogenase which converts

PG to 15-keto-PG, and (2) a 13,14-reductase which converts 15-keto-13, 14-dihydro-PG to the i3,14-dihydro-PG, (8).

Vane has proposed that the

physiological significance of the lung's degradative system for circulating PG is to protect the systemic arterial circulation from the high concentration of PG that may exist in the systemic venous blood. However, recent reports have indicated that the PG metabolites themselvesmay have biological a c t i v i t y similar to the PG (9,10). The purpose of the present communication is several fold. Since the enzymes responsible for the enzymatic conversion of PG are intracellular, somemechanism(s) must exist for the selective removal of PG from the circulation. Using an isolated perfused rat lung preparation we have investigated the mechanism(s) responsible for the selective removal of PG from the circulation by the lung. Since the PG metabolites may have biological a c t i v i t y , the effects of time and pulmonary arterial blood concentration of PG on the appearance of PG metabolites in the pulmonary venous effluent were examined.

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MATERIALS AND METHODS

Materials: 3H-PGF~ (9-3H), 3H-PGE,(5,6-3H), 3H-PGAI(5,6-3H) and 3H-PGBI(5,6-3H), "C-dextran (M.W. ~ 70,000), and Aquasol s c i n t i l l a t i o n cocktail were purchased from New England Nuclear, Boston, Mass. 3H-15keto-PGF2a was synthesized from 3H-PGF2a by the method of Levine and Gutierrez-Cernosek ( I I ) . Authentic standards of PGF2a -Tham, PGEI, PGE2, 15-keto-PGF2a, 15-keto-13,14-dihydro-PGF2a, 13,14-dihydro-PGF2a, 15-keto-PGE I, 15-keto-13,14-dihydro-PGE 1 , 13,14-dihydro-PGE 1 , 15-ketoPGAl, 15-keto-13,14-dihydro-PGA 1 and 13,14-dihydro-PGA 1 were gifts from Dr. John Pike, Upjohn Co., Kalamazoo, Mich. Silica gel G thin layer plates (250 u ) were purchased from Analab Inc., Wilmington, Del. B-NAD+ was obtained from Sigma Chemical Co., St. Louis, Mo. Blue dextran (M.W. " 2xlO 6) was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). Perfusion of Rat Lungs The lung perfusion technique was a modification of the method previously described (12,13) for the rabbit lung. Briefly, the lung perfusion system consists of an a r t i f i c i a l thorax, a perfusate supply and an a i r supply. pressure.

The lung was ventilated by an alternating negative

The circulatory system was designed with two open reservoirs

connected by a 3-way stopcock to the pulmonary artery.

Effluent from

the lung was collected via the pulmonary vein in a fraction collector (Gilson Medical Electronics, Inc., Middleton, Wisconsin). In addition, an injection port was connected to the pulmonary artery allowing direct bolus injection of chemicals into the pulmonary artery. Flow rates were measured by a flow-transducer and meter (Biotronics Lab, In~., Silver Springs, Maryland). Rats (Sprague Dawley-CD, purchased from Charles River, Wilmington, Mass.) weighing approximately 200-250g were anesthetized with halothane, the pulmonary artery cannulated in situ, the lungs removed and then perfused with a medium consisting of the following: NaCl, l l 8 mM; KCI, 4.75B~M; KH2PO4, 1.19mM; MgSO4, 1.19mM; CaCl2, 2.54mM; NaHCO3, 25n$I; glucose, 5mM and 4.5% bovine serum albumin. Perfusate was adjusted to pH 7.4, equilibrated with 5% CO2 in oxygen and maintained at 37°C. Lungs were perfused with this f l u i d at a rate

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of either 30ml/min. 6r 60 ml/min. Before the i n i t i a t i o n of the experiment, the success of perfusion was assessed by injection of blue dextran into the pulmonary artery. absence of color.

Non-perfused areas were noted by

At the end of the experiments, lungs were weighed

and edematous lungs (greater than I0% increase in weight) discarded. 3H-PG and the vascular marker 14C-dextran was injected into the. pulmonary artery of IPL as a bolus, or the lungs were perfused with a medium containing 3H-PG and 14C-dextran at a fixed arterial perfusate concentration. Effluent from the lung was collected and aliquots mixed with Aquasol s c i n t i l l a t i o n cocktail. Both 3H and 14C were measured in each sample and corrected for spillover and quenching. Calculation of Net Rate of Accumulation of Radioactivity by Lung From a knowledge of the radioactivity in the arterial perfusate and the radioactivity in the effluent samples, the net rate of accumulation by the lung of either 3H or 14C during any time interval was determined as follows: Net Rate of Uptake = Effluent Sample Volume x (Arterial Conc.-Venous Conc.) Duration of Sample Collection Detection of Metabolites in the Effluent from the IPL Lung effluents containing 3H-PG and their metabolites were acidified to pH-3.5 with l N HCI and then extracted with EtAC. The EtAC was evaporated to dryness under vacuum and the residue dissolved in MeOH. Aliquots of the MeOHextract were applied to s i l i c a gel G plates and the plates were developed using the following solvent systems: (a) benzene, EtOH, acetic acid (45:4.5:0.5) for PGAl (b) benzene, dioxane, acetic acid (130:24:4) for PGEl and (c) CHCl3, MeOH, acetic acid, H20 (180:17:2:1.3) for PGF2 . After development the plates were dried and scraped in sections (.3 - .5cm) into s c i n t i l l a t i o n vials containing dioxane-based s c i n t i l l a t i o n cocktail.

Radioactivity was measured using

liquid s c i n t i l l a t i o n techniques. Authentic standards were visualized by spraying the plates with 5% phosphomolybdic acid in EtOH and heating at llO°C.

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Metabolism of PG by Pulmonary Homogenates Rats were sacrificed by cervical dislocation.

The lungs were

removed and homogenized in O.l M phosphate buffer, pH-7.4 (tissue to medium ratio, l:lO). 3H-PGF2~, 3H-PGEI, 3H-PGAI, or 3H-PBGI at a concentration of l.O nmole/ml was incubated with 0.5 ml of the lung homogenate as described by Parkes and Eling (14).

After acidification of

the incubation mixture to pH-3.5 with l N HCl, PG and their metabolites were extracted with EtAC and analyzed by thin layer chromatography as described in the previous section.

RESULTS Bolus Injections After a bolus injection of .006 nmoles of 3H-PGF2e and the vascular marker 14C-dextran into the pulmonary artery, the effluent from the pulmonary vein was collected at l . l second intervals.

Selected samples

of the effluent were studied by thin-layer chromatography and a maximum of three peaks were observed in any one sample. These peaks corresponded to PGF2~, 15-keto-PGF2~(M-I ) and 15-keto-13,14-dihydro-PGF2~ (MII), and were identified by comparison to authentic standards. The appearance of these metabolites and unchanged PGF2e in the effluent as a function of time after the bolus injection is shown in figure I. The peak for MII was displaced by approximately lO seconds from the vascular marker dextran, that for MI approximately 2-3 seconds whereas the peak for the unchanged PGF2~ was identical to that of dextran (figure la). More than 95% of the tritium from the injected dose of 3H-PGF2e was recovered in the effluent in 80 seconds and MI represented 50 per cent of the dose, MII 37 per cent, and PGF2e 13 per cent. I t was shown that dextran, at the concentrations used in these studies, had no effect on the uptake of PG. Analysis of the effluent for total radioactivity after a bolus injection of 0.004 or 0.4 nmoles of either 3H-PGB or 3H-PGA, together with 14C-dextran showed that the tritium peak coincided with the dextran peak (figure Ib).

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In each case, only one peak was observed in thin-layer

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chromatographic analysis and this peak corresponded to the parent prostaglandin, PGBl , or PGAI. The non-removal of PGAl or PGBl from the vasculature implied that the cells of the lung are impermeable to these compounds and that the structurally very similar PGF2~must be accumulated i n t r a c e l l u l a r l y by some mechanism other than diffusion or binding. Rates of Removal of Prostaglandin From Vasculature into Lung In order to study the removal mechanisms, the measurement of the unidirectional f l u x of PG from the perfusate into the lung and an examination of this f l u x as a function of perfusate concentration was desirable.

To accomplish this, a constant arterial perfusate concentra-

tion, CA, was presented to the lung and the total radioactivity in the venous effluent determined as a function of time. The open circles in figure 2 show the net rate of uptake of tritium from the perfusate into the lung as a function of time after introduction of perfusate containing 3H-PGEI. Before the vascular space is completely perfused with radioactivity, there w i l l be an apparent uptake of tritium due to dilution into the vascular space.

In order to correct for this

a r t i f a c t , the net rate of uptake of the vascular marker 14C-dextran was also determined. Since the apparent uptake due to dilution into the vascular space is the same for tritium and 14C-dextran, the difference between the net rate of uptake of tritium and 14C-dextran w i l l be a true measure of the net rate of uptake of tritium from the vascular space into the lung. The closed circles in figure 2 represent the difference between the net rate of uptake of tritium and 14C-dextran. Since there are different vascular pathlengths in the lung between the pulmonary artery and the pulmonary vein, the difference velocity (solid circles) does not peak until a l l vascular space is completely perfused with radioactivity.

This time at the peak in the difference velocity was

assumed to be the "zero time", and for a perfusate flow rate of 30ml/min i t varied between 7 and I I seconds. After this zero time was reached, there was no further apparent uptake of 14C-dextran and the two velocities became equal (open and solid circles in figure 2).

The decay in the net

rate of uptake of tritium was then due to the efflux from the lung into

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the perfusate of PGEl and/or i t s metabolites.

The difference velocity

(solid circles, figure 2) at "zero time" was a measure of the unidirectional flux or the uptake velocity of PGEl from the perfusate into the lung.

At

this time l i t t l e or no radioactivity had effluxed from the lung into the perfusate. The unidirectional f l u x of PGEl was examined as a function of CA over the range 0.05 to 20.0 nmoles/ml at a perfusion flow rate of 30ml/min.

Figure 3a shows that the f l u x was saturable with respect to

the supply rate [SR = CA X flow rate].

The straight line obtained with

the Woolf plot in figure 3b verifies that this relationship was a rectangular hyperbola with a maximum uptake velocity of 2.6 nmoles/sec. and a supply rate of 3.23 nmoles/sec,required to give a half maximum uptake velocity.

This type of relationship between the f l u x and the

supply rate (or CA) suggests that a carrier-mediated process was involved in the removal of PGEl by the lung.

We also examined the rates of

removal of PGF2~, PGBl , PGAl , and 15-keto-PGF2~ from the vasculature into the lung. The rate of removal of PGF2~ from the vasculature into the lung also appeared to be saturable with respect to perfusate concentration; however, i t was impossible to cover the concentration range necessary for the proper analysis of the uptake velocity versus CA curve since perfusate concentration of PGF2~ above l.O nmole/ml caused a reduction in the perfusate flow rate.

No detectable rate of removal by the IPL was

observed for PGAl , PGBl and 15-keto-PGF2~ over the perfusate concentration range of .OOl to l.O nmoles/ml. Measurement of the rate of removal of PGEl or PGF2~ by the lung from the vasculature at a perfusate flow rate of 60 ml/min, indicated that the relationship between the uptake velocity and SR was the same as that established at a flow rate of 30 ml/min. (figure 3a). open circles in figure 3a i l l u s t r a t e this for PGEI.

The

Thus the uptake

of PGEl appeared to be dependent on the rate at which the PGEl was supplied to the lung rather than on i t s concentration in the perfusate.

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Examination of Metabolites in Effluent Figures 4a and 4b show the concentration of PGF2~ and the two metabolites, 15-keto-PGF2~ (MI) and 15-keto-13,14-dihydro-PGF2~ (MII) in the effluent as a function of time after the i n i t i a t i o n of a constant arterial perfusate concentration. Over a concentration range of .005 to 2.0 nmoles/ml of PGF2: in the perfusate, MI and MII were the only metabolites detected by thin layer chromatographic analysis.

The concentration of PGF2~, MI and MII in

the effluent increased with time until a plateau value was reached (figures 4a and 4b).

The ratio of the plateau values for MI to MII

was dependent on the supply rate of PGF2~ (figure 5).

The increase in

the ratio to a value greater than one implies that the PG dehydrogenase (PGF2~ + MI,) has a larger maximum conversion rate than the 13,14reductase, (MI + MII). During perfusion with a constant CA of PGEl , the metabolites 15keto-PGEl and 15-keto-13,14-dihydro-PGEl were detected in the effluent by thin layer chromatography analysis.

We could not detect 13,14-

dihydro-PGEl in the effluent. The appearance in the effluent of PGEl and i t s metabolites was similar to that of PGF2~ and i t s metabolites (figures 4a and 4b).

The variation of the ratio of the plateau values

of 15-keto-PGEl to l~,14-dihydro-15-keto-PGE l with the SR was similar to the corresponding ratio of the metabolites of PGF2~ (figure 5). During a perfusion with a constant SR, .OOl or l.Onmoles/sec.of either PC~A l , PGBl or 15-keto-PGF2~, no metabolites of these PG's were detected by thin layer chromatography. Calculation of the Net Rate of Removal or the Metabolism Velocity for PGF2~ and PGEl The net rate of removal of PGF2~ or PGE1 from the perfusate by the lung can be calculated from the effluent concentration of the PG: net rate = (CA - effluent concentration) X flow rate.

This net rate or

the metabolism velocity is the difference between the rate of removal of PG from the perfusate by the lung, (figure 2) and the rate of efflux

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of any unmetabolized PG from the lung into the perfusate.

I f all of the

PG that was removed from the perfusate by the lung was metabolized, then the net rate should equal the uptake velocity.

For the range of SR for

PGF2~ of O.Ol to l.O nmoles/sec., and for PGEl of O.Ol to lO.O nmoles/sec., the net rates were calculated after the PG concentration in the effluent had plateaued (figures 4a, 4b).

Figure 6 is a plot of the net rates

against the corresponding uptake velocities (figure 3).

The uptake

velocity was equal to the net rate for SR of less than O.l nmoles/sec., which corresponds to uptake velocities less than 0.08 nmoles/sec, for PGEl and PGF2 .

At higher supply rates the uptake velocity was greater

than the net rate.

These results imply that the IPL was able to metabolize

all of the PG that was removed from the perfusate by the lung's uptake mechanism for a l l SR of less than O.l nmoles/sec., whereas at all higher SR, some of the PG accumulated by the cells effluxes unmetabolized into the perfusate.

This conclusion is consistent with the distinct difference

in the time required for the effluent given of PGF2e to reach the plateau values in figures 4a and 4b.

For the lower SR (figure 4a), the effluent

concentration of PGF2a plateaued immediately, often within 12 seconds, whereas at the higher SR (figure 4b), the effluent concentration of PGF2~ does not plateau until much later.

The increase in effluent

concentration with time shown in figure 4b was due to the efflux from the lung into the perfusate of unmetabolized PG which had accumulated in the lung's cells. The rate of the enzymatic conversion of PGEl or PGF2~was equal to the rate of removal after the plateau concentration was reached (figures 4a and 4b).

This follows since the rate of efflux of the metabolites must

be equal to the enzymatic conversion rate, and the net rate is equal to the rate of efflux of metabolites after steady state conditions were obtained.

Figure 6 is a plot of the metabolism velocity versus the

corresponding uptake velocity. Percentage of Supply Rate that is Removed and Metabolized The percentage of the SR that was removed from the perfusate by the lung is % = Uptake Velocity SR

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Figure 7, open circles, is a plot of this percentage versus the supply rate for PGEI.

The percentage of the supply that was metabolized is % = Metabolism Velocity X lO0 SR

Figure 7, closed circles, is a plot of this percentage versus the SR for PGEI.

The two percentages are equal at the lower SR of less than

O.l nmoles/sec., but as the SR increases the percentage that was metabolized decreases much more rapidly than the percentage that was removed. This occurs at the higher SR since some of the PG that was accumulated by the removal mechanism effluxes unaltered from the lung into the perfusate.

Relationships similar to those in figure 7 were obtained

for PGF2. Inhibition Studies Table one shows the effect of the presence of PGEl and PGE2 in the perfusate on the uptake velocity and metabolism of 3H-PGF2~ at a SR = O.l nmoles/ml, and the effect of the presence of PGE2 and PGF2~ on the uptake velocity and metabolism of 3H-PGEI at a SR = O.l nmoles/ml. The percentage inhibition of the uptake velocity or the percentage inhibition of the metabolism increased as the concentration of the i n h i b i t o r increased.

In each case, the percentage inhibition of the

metabolism was greater than the percentage inhibition of the uptake velocity.

This is consistent with the earlier observation that the

removal system has a greater capacity than the metabolism system (figure 7).

PGBl at a lO-fold higher concentration had no effect on the

uptake velocity or metabolism of 3H-PGEI or 3H-PGF2 . Figure 8 shows the effect of PGE2 at a SR = 2.5 nmoles/sec, on the uptake velocity of 3H-PGEI over a SR range of O.l to lO.O nmoles/sec. In the presence of PGE2, the maximum uptake velocity of PGEl decreased from the control value of 2.6 ± .2 to 2.2 ± 0.2 nmoles/sec, and the SR necessary to give half-maximum uptake velocity increased from the control value of 3.2 ± .4 to 7.0 ± .2 nmoles/sec. The two-fold increase in the Km in the presence of the PGE2, and the similarity in the maximum uptake velocities in the presence and absence of the PGE2 implies that PGEl

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and PGE2 are competing for the same transport or removal process. Metabolism of PG by Pulmonary Homogenates PGF2a, PGEl , PGAl , and PGBl at a concentration of l.O nmole/ml were incubated for 15 minutes with lung homogenates from rat lung. All of the prostaglandins were degraded by lung homogenates except PGBl which is in agreement with previous workers (15).

With these experimental

conditions, 64 ± 5 percent of PGAl , 60 ~ 8 percent of PGF2a, and 87 ~ 4 percent of PGEl were degraded. Thus, the lack of PGAl metabolism by the IPL may be the result of the inaccessibility of PGAl to the sites for i t s enzymatic degradation. DISCUSSION Evidence which suggests that the lung's prostaglandin inactivation system consists of a transport system in addition to the intracellular dehydrogenase and reductase enzymes is as follows: (1)

The kinetic similarity in behavior between PGAl or PGBl and

the vascular marker dextran (figure Ib) indicates that very l i t t l e PG are accumulated i n t r a c e l l u l a r l y by diffusion. (2)

The saturability of the uni-directional f l u x of PGEl (PGF2a)

into the lung with respect to the arterial perfusate concentration (figure 3) suggests a transport process. (3)

The inhibition of the uni-directional f l u x of one PG by another

(table l ) and the concentration dependence of this inhibition (figure 8) also suggest a transport system. PG may cross the cellular membrane by diffusion to a limited extent, but the system primarily responsible for the high percentage removal of PG from the circulation by the lung is a transport system. Examinations of transport systems require measurements of unidirectional fluxes.

Uni-directional fluxes in perfused organs can be

measured by extrapolation of the net rate of removal of radioactivity to zero time (figure 2).

Using this technique, Iverson et al examined

the transport system for catecholamines in the isolated perfused heart

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(16) and our laboratory investigated the 5-HT transport system in the isolated perfused lung (13).

The uni-directional f l u x can also be

determined from the difference in arterial and venous concentration of the chemical when there is no efflux of unmetabolized chemical which has been accumulated by the tissue.

At low perfusate concentrations

this condition is f u l f i l l e d in the present study, and, thus, identical values for the uni-directional f l u x were obtained (figure 6).

A more

detailed discussion of these two techniques for the measurement of the uni-directional f l u x of chemicals from the perfusate into the lung has been published (13). The specificity of the inactivation system for the E and F type PG in cat and dog has been previously established (5,6). show that the same selectivity exists in the rat.

Our results

The metabolism of

PGAl by the I00,000 x g supernatant fraction of rat lung tissue in a manner quantitatively similar to that of PGEl and PGF2~ and the i n a b i l i t y of the lung to metabolize circulating PGAl implies that the specificity is due to the transport system and not the metabolic system.

I t appears

that this selectivity of the transport system does not exist in the lung of the guinea pig (3).

A detailed examination of the structural require-

ment of PG type compounds for the lung's inactivation system would be desirable since the plasma half-lives of the various PG analogues being considered as drugs would be determined in part by the inactivation system. PG are removed from the circulation and inactivated by this pulmonary system with a high degree of efficiency

equal to 80% for supply rates

less than 0.05 nmoles/ml. This is consistant with results reported by previous investigators (3-7).

However, i t has not been previously shown

that the efficiency of the pulmonary system rapidly declines with increasing SR. The a b i l i t y of the intracellular pulmonary enzymes to metabolize PG decreases more rapidly than the a b i l i t y of the transport system to remove the PG from the circulation. Thus, removed PG are returned unaltered to the general circulation.

The mechanism by which

the PG and PG metabolites return to the general circulation is not known. The rapid decline in the efficiency of the PG inactivation system with SR can be of importance in some diseased states such as medullary carcinoma of the thyroid (17) and the carcinoid syndrome (18), when

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abnormally high concentrations of PGE are found in the peripheral venous circulation.

I f the pulmonary arterial concentration of PGE was

s u f f i c i e n t l y elevated significant amounts of unaltered PGE would excape from the lung into the systemic arterial circulation. Crutchley and Piper ( l l ) showed that various chemicals could i n h i b i t guinea pig lung's PG inactivation system. This inhibition could result from effects on either the transport system and/or the metabolic system. The PG antagonists polyphloretin phosphate (PPP) and diphloretin phosphate (DPP) were potent inhibitors of the inactivation system at concentrations less than that required for antagonistic actions.

DPP and PPP are

probably inhibiting the transport system since they exist in a highly ionized form at physiological pH which would make intracellular penetration d i f f i c u l t .

Our laboratory has shown that in guinea pigs exposed to

I00% 02 for 48 hours or to 8 ppm NO2 for 6 hours, there is a decrease, 50% for 02 and 60% for NO2, in the lung's PG dehydrogenase a c t i v i t y (20, 21).

This w i l l obviously affect the inactivation system. Naito and

G i l l i s (22) showed that the anesthetics, halothane and nitrous oxide inhibited the lung's inactivation system for noradrenaline.

These studies

indicate that further investigations of the effects of chemicals in the circulation and of inhaled pollutant gases on the transport and metabolic components of the lung's inactivation system for PG could be important. In particular, the cardiovascular effects of chronic and acute exposure to environmental pollutants such as NO2, SO2, and ozone could in part be due to their effects on the lung's PG inactivation system. Although numerous studies have shown that PG are metabolized by the lung, our results are the f i r s t to show the time pattern of the appearance of metabolites in the effluent and relative effluent concentrations of the metabolites as a function of the arterial concentration of PG. The nature of the PG metabolites and their relative concentrations in the pulmonary venous effluent are of considerable interest since recent work indicates that these PG metabolites have similar or greater biological a c t i v i t y than the parent PG (9, lO).

In view of these findings, the uptake and

metabolism of PG by the lung may not result in a biological inactivation with a l l PG and in all species.

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Bito and co-workers have suggested that PG transport systems exist in several tissues such as choroid plexus, anterior uvea, kidney cortex, and vagina and that these systems are substrate specific and energydependent (23, 24).

Using a bladder-like preparation of the isolated

rabbit vagina, Bito showed that PG would cross the membrane in only one direction (24).

Our results are the f i r s t attempt to measure the

uni-directional f l u x of PG across a membrane and to determine this f l u x as a function of PG concentration in the medium. Thus, at present, we cannot compare our transport parameters (Km, Vmax) with those of other investigators. Bito and Baroody (25) have reported that E and F type PG are excluded from red blood cells whereas PGA is accumulated by these cells.

A transport system may exist in red blood cells for the A type

PG but not for PGE and PGF. This selectivity would be different than that found in the rat lung.

The exclusion of the E and F type PG by

the red blood cells is consistent with our findings that PG cannot freely diffuse across cellular membranes. The existence of PG transport systems may have important biological significance.

PG transport systems could be involved in the mechanisms

associated with the biological actions of the PG and the regulation of intracellular concentrations.

The transport system in the lung may

regulate arterial blood concentrations of PG by removing them from the venous blood and may prevent the PG that are synthesized by the lung during normal and patho-physiological conditions, such as embolism and anaphylactic shock,from reaching the arterial blood.

Bito (24) proposed

that transport systems exist in the choroid plexus and anterior uvea for the purpose of removing PG from the extracellular fluids of brain and eye, thereby preventing elevated levels of PG in these tissues.

Lewis

and Piper (26) have recently proposed that a transport system exists in the plasma membrane of adipose cells and that the i n t r a c e l l u l a r l y synthesized PG are transported from the inside of these cells and released into the extracellular space by means of this system.

In addition, these

workers proposed that anti-inflammatory steroids may act by inhibiting this system; however, this concept was not substantiated by the results of other workers (27, 28, 29)

Thus, further studies are necessary to

obtain an understanding of PG transport systems and their role in biological actions of PG.

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PROSTAGLANDINS Figure l.A

The Appearance of 14C-Dextran, 3H-PGF2~and Metabolites in the Venous Effluent from the IPL after a Bolus Injection into the Pulmonary Artery of. IPL

On the vertical axis is the normalized dpm/ml of effluent for each compound; on the horizontal axis is the time (seconds) after the bolus injection. The dpm/ml was normalized with respect to the peak height of dextran. 14C-dextranm,3H-PGF2~ e , 3H-15-keto-PGF2~ O, 3H-15-keto-13114-dihydro-PGF2~ A . Figure l.B

The Appearance of 14C-Dextran and 3H-PGBI or 3H-PGAI in the Venous Effluent from the IPL after a Bolus Injection in.to the Pulmonary Artery of IPL

On the vertical axis is the normalized dpm/ml of effluent; on the horizontal axis is the time (seconds) after the bolus injection. Dpm/ml was normalized with respect to the peak height of dextran. 14C-dextran| ,

3H-PGB1 or 3H-PGA10. Figure 2.

The Variation with Time of the Net Rate of Uptake of Radioactivity from Perfusate into the IPL

The open circles are the net rate of uptake of tritium from the circulation into the lung. The solid circles are the difference in the net rate of uptake between tritium and 14C-dextran. On the horizontal axis is the time (seconds) after the initiation of a constant arterial concentration of 3H-PGEI (0.30 nmoles/ml) and 14C-dextran. The arrow denotes the time when the difference in the rates between tritium and 14C-dextran uptake was maximum. Lungs were perfused at approximately 30 ml/min.

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PROSTAGLANDINS

Figure 3.

The Dependence on the Supply Rate of the Uptake Velocity of PGEl into the IPL

(a) On the vertical axis is the uptake velocity (nmoles/sec.) for PGEI; on the horizontal axis is the supply rate (nmoles/sec.). The solid circles are the uptake velocities determined at a flow rate of 30 ml/min.; the open circles are uptake velocities measured at a flow rate of 60 ml/min.

(b) Woolf Plot. On the vertical axis is the ratio of the supply rate to the uptake velocity; on the horizontal axis is the supply rate (nmoles/sec.). The solid circles are data obtained at 60 ml/min, flow rate. The straight line was obtained by a linear regression analysis. The kinetic parameters obtained were Km = 3.2 ~ 0.4 nmoles/sec, and Vmax = 2.6 ~ .2 nmoles/sec./ organ.

Figure 4.

The Appearance of 3H-PGF2~ and Metabolites in the Venous Effluent from IPL During Perfusion with a Constant Arterial Concentration of 3H-PGF2a

On the vertical axis is the concentration of PGF2~ and metabolites in the IPL effluent (nmoles/ml). On the horizontal axis is the time (seconds) after i n i t i a t i o n of the constant concentration of 3H-PGF2 . (3H-PGF2~ = O, 15-keto PGF2~ = O, 3H-15-keto-13,14-dihydro-PGF2~ = A). Lungs were perfused at approximately 30 ml/min.

660

(a)

Lungs perfused with 3H-PGF2~ at 0.022 nmoles/ml.

(b)

Lungs perfused with 3H-PGF2~ at 2.01 nmoles/ml.

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Figure 5.

The Effect of Supply Rate on the Concentration of 3H-PGF2~ tT2i'E¢l~ill,{:}.-lmi lit ~i?~t:~tm n.-lmi l l s ~ i ' i ' ; ~

il'~ I $

On the vertical axis is the ratio of the concentration in venous effluent of 15-keto-PGF2~ (MI) to 15-keto-13,14-dihydro-PGF2a (MII); on the horizontal axis is the supply rate of 3H-PGF2~ (nmoles/sec.)

Figure 6.

A Comparision of Uptake Velocity to Net Rate of PG Removal or Metabolism Velocity in the IPL

On the vertical axis is the net rate of PG removal or metabolism velocity (nmoles/sec.); on the horizontal axis is the uptake velocity (nmoles/sec.).

Figure 7.

Solid circles refer to PGEl , open circles are PGF2~ .

The Effect of Supply Rate on the Percentage Removal and Metabolism of PGEl by IPL

On the vertical axis is the percentage removal or percentage metabolism of PGEI.

On the horizontal axis is the supply rate (nmoles/sec.).

The

solid circles are percentages of PGEl metabolized; the open circles are percentages of PGEl removed from the circulation by IPL.

Figure 8.

The Effect of the Presence of PGE2 on the Uptake Velocity of 3H-PGEI into the IPL

Lungs were perfused with a mixture of 5 nmoles/ml, of PGE2 and varying concentrations of 3H-PGEI at a flow rate of approximately 30 ml/min.

Data is plotted in the form of Woolf plot.

On the vertical

axis is the ratio of the supply rate of PGEl to the uptake velocity. the horizontal axis is the supply rate of PGEl (nmoles/sec.). are experimental data obtained in the presence of PGE2.

APRIL

1976

V O L . 11 N O . 4

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The circles

The dashed lines

661

PROSTAGLANDINS

are control data obtained from figure 3.

The following kinetic constants

were obtained by linear regression analysis:

3H-PGEI, Km = 3.2 ± 0.04

nmoles/sec., Vmax = 2.6 ~ 0.2 nmoles/sec.; 3H-PGEI + 5 nmoles/ml PGE2, Km = 7.0 C 0.2 nmoles/sec., Vmax = 2.2 ± 0.2 nmoles/sec.

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Table I Inhibition of the uptake velocity and metabolism of 3H-PG by the IPL in the presence of a second PGa

Substrate (O.l nmole/ml)

3H-PGF2~

Inhibitor (nmoles/ml)

% Inhibition of uptakevelocity (X + S.D.)

% Inhibition of metabolism (X +_ S.D.)

PGEl (lO)

49 ~ 2.6 b

lO0

PGEl (5) PGE] (I.6)

26 ± 3.9 0

lO0 80 ~ 4

3H-PGF2~

PGE2 (lO) PGE2 (5) PGE2 (1.6)

60 ~ l 36 ~ 2.8 0

lO0 lO0 95 ~ 8

3H-PGEI

PGE2 (I0) PGE2 (5) PGE2 (I.6)

64 ± 3 44 ~ 2 13 ~ 2

I00 lO0 77 ~ 6

3H-PGEl

PGF2~(I.6)

8.3 ~ 6.7

51 ~ 5

a Lungs were perfused with a mixture of the inhibitor PG at a concentration indicated in the parentheses, and the substrate PG at a concentration of O.l nmoles/ml. b Each result is the X +- S.D. of three experiments.

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REFERENCES I.

Fanburg, B. L. Prostaglandins and the Lung, Amer. Rev. of Resp. Dis. 108:482-489, 1973.

2.

Oesterling, T. 0., Morozowich, W., Roseman, T. J. J. of Pharm. Sci. 61: 1861-1895, 1972.

3.

Piper, P. J., Vane, J. R., Wyllie, J. H. by the Lungs, Nature 225: 600-604, 1970.

4.

Ferreira, S. H., Vane, J. R. Prostaglandins: Their Disappearance from and Release into the Circulation, Nature 216: 868-873, 1967.

5.

McGiff, J. C., Terrangno, N. A., Strand, J. C., Lee, J. B., Lonigro, A. J. Selective Passage of Prostaglandins Across the Lung, Nature 223: 742-745, 1969.

6.

Roberton, R. P. Differential in vivo Pulmonary Degradation of Prostaglandins El , Bl , and Al,~e~'~. J. Phys., 228: 68-70, 1975.

7.

Hook R., G i l l i s , C. N. The Removal and Metabolism of Prostaglandin El by Rabbit Lung, Prostaglandins 9: 193-201, 1975.

8.

Anggard, E. Studies on the Analysis and Metabolism of the Prostaglandins, Ann. New York Acad. Sci. 180: 200-217, 1971.

9.

Jones, R. L. Actions of Prostaglandins on the Arterial System of the Sheep: SomeStructure-Activity Relationships, Proc. Br. Pharmac. Soc., 1975, p. 464.

Prostaglandins,

Inactivation of Prostaglandins

lOo Dawson, W°, Lewis, R. L., McMahon, R. E., Sweatman, W. J. F. Potent Bronchoconstrictor Activity of 15-keto Prostaglandin F2~, Nature 250: 331-332, 1974. If.

Levine, L., and Gutierrez-Cernosek, R. M. Preparation and Specificity of Antibodi.es to 15-keto-Prostaglandin F2a, Prostaglandins 2: 281-294, 1975.

12.

Orton, T. C., Anderson, M. W., Pickett, R. D., Eling, T. E., Fouts, J. R. Xenobiotic Accumulation and Metabolism by Isolated Perfused Rabbit Lungs, J. Pharm. Exper. Ther. 186: 482-497, 1973.

13.

Pickett, R. D., Anderson, M. W., Orton, T. C., Eling, T. E. The Pharmacodynamics of 5-Hydroxytryptamine Uptake and Metabolism by the Isolated Perfused Rabbit Lung, J. Pharm. Exper. Ther. 194: 545-553, 1975.

14.

Parkes, D. G., Eling, T. E. Characterization of Prostaglandin Synthetase in Guinea Pig Lung. Isolation of a New Prostaglandin Derivative from Arachidonic Acid, Biochemistry 13: 2598-2604, 1975.

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15.

Nakano, J., Anggard, E., Samuelsson, B. 15-Hydroxy-Prostaglandin Dehydrogenase. Prostaglandins as Substrates and Inhibitors, Eur. J. Biochem. l l : 386-389, 1969.

16.

Iversen, L. L. The Uptake of Catecholamines at High Perfusion Concentrations in the Rat Isolated Heart: A Novel Catecholamine Uptake Process, Brit. J. Pharmacol. 25: 18-33, 1965.

17.

Williams, E. D., Karin, D. M. M., Sandler, M. Prostaglandin Secretion by Medullary Carcinoma of the Thyroid: A Possible Cause of the Associated Diarrhea, Lancet l : 22-23, 1968.

18.

Jaffe, B. M., Behrman, H. R., Parker, C. W. Radioimmunoassay Measurement of Prostaglandins El , Al and F in Human Plasma, J. Clin. Invest. 52: 398-405, 1973.

19.

Crutchley, D. J., Piper, J. Prostaglandin Inactivation in GuineaPig Lung and Its Inhibition, Br. J. Pharmac. 52: 197-203, 1974.

20.

Parkes, D. G., Eling, T. E. The Influence of Environmental Agents on Prostaglandin Biosynthesis and Metabolism in the Lung, Biochem. J. 146: 549-556, 1975.

21.

Parkes, D. G., Eling, T. E., Anderson, M. W. Unpublished observation.

22.

Naito, H., G i l l i s , C. N. Effects of Halothane and Nitrous Oxide on Removal of Norepinephrine from the Pulmonary Circulation, Anesthesiology 39: 575-580, 1973.

23.

Bito, L. Z. Accumulation and Apparent Active Transport of Prostaglandins by Some Rabbit Tissues in v i t r o , J. Physiol. 221: 371-387, 1972.

24.

Bito, L. Z. Saturable, Energy-Dependent, Transmembrane Transport of Prostaglandins against Concentration Gradients, Nature 256: 134-136, 1975.

25.

Bito, L. Z., Baroody, R. The Impermeability of Rabbit Erythrocytes to Prostaglandins, Am. J. Physiol. 228, 1975.

26.

Lewis, G. P., Piper, P. J. Inhibition of Release of Prostaglandins as an Explanation for Someof the Actions of Anti-lnflammatory Corticosteroids, Nature 254: 308-311, 1975.

27.

Gryglewski, R. J., Panczenko, B., Korbut, R., Grodzinska, L., and Ocetkiewicz, A. Corticosteroids Inhibit Prostaglandin Release from Perfused Mesenteric Blood Vessels of Rabbit and From Perfused Lungs of Sensitized Guinea Pig, Prostaglandins lO: 343-355, 1975.

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28.

Kantrowitz, F., Robinson, D. R., and McGuire, M. B. Corticosteroids Inhibit Prostaglandin Production by Rheumatoid Synovia, Nature 258: 237-239, 1975.

29.

Tashjian, Jr., A. H., Voelkel, E. F., and McDonough, J. Hydrocortisone Inhibits Prostaglandin Production by Mouse Fibrosarcoma Cells, Nature 258: 239-240, 1975.

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