Work in Progress
INHIBITION OF PULMONARY PROSTAGLANDIN METABOLISM BY INHIBITORS OF PROSTAGLANDIN BIOTRANSPORT (PROBENECID AND BROMCRESOL GREEN).
L. Z. Bito and R. A. Baroody
Ophthalmology Research, Columbia University College of Physicians and Surgeons, New York, N.Y. 10032
The transfer of prostaglandins (PGs) across the blood-brain (I) and blood-aqueous barriers (2), and their absorption from the lumen of the rabbit vagina (3) is mediated by saturable, "carrier-mediated", transport processes.
In vitro studies demonstrated that such a PG transport
process can take place against a concentration gradient (4) and thus it may, under certain conditions, represent an active transport process. The physiological significance of PG transport processes has been underscored by the observation that rabbit red blood cells, and thus presumably the basic cell membrane in general, are impermeable to PGs (5,6).
These observations indicate that the flux of PGs across some
specialized membranes is mediated by special mechanisms rather than being accounted for by simple diffusion. An earlier survey of concentrative PG accumulation by animal tissues (7,8) suggested that in addition to the chorold plexus, ciliary processes and kidney cortex, the mammalian lung also possesses such PG transport mechanisms.
Since the key enzymes in pulmonary PG metabolism
must be intracellular, the effective metabolism of PGs during their passage through the lungs must require an initial step of carriermediated transmembrane transport (7,9). The extent of 3H accumulation by the rat lungs, following shortterm intravenous infusion of 3H-PGF2a, is drastically reduced in animals which were pretreated with a PG transport inhibitor (i0). Experiments currently in progress show that two PG transport inhibitors, probenecid and bromcresol green, both inhibit the metabolism of PGF2a by the isolated perfused rat lung.
One of these substances, namely bromcresol green,
has no inhibitory effect on the metabolism of this PG by the highspeed supernatant fraction of lung homogenates.
Io-15-75
PROSTAGLANDINS OCTOBER 1975
VOL. 10 NO. 4
633
PROSTAGLANDINS
Columbla-Sherman Chloropent
albino rats (400-450 gm) were anesthetized with
(Fort Dodge Laboratories,
Inc., Fort Dodge, Iowa).
The heart
was exposed and heparln (500 units), in saline, was injected into the right ventricle.
The heart and lungs were removed and a cannula was
inserted into the pulmonary artery.
This preparation,
suspended in a
moist chamber maintained at 37°C, was infused at a rate of 8 ml/min with Kreb's solution gassed with 95% 02 and 5% CO 2.
After 15 min the in-
fusion solution was changed to one containing I00 ng/ml of PGF2~ and three effluent samples were collected at I mln intervals.
The PGF2a was
kindly supplied by Dr. John E. Pike of The Upjohn Company (Kalamazoo, Mich.).
After this initial control perfusion the infusion solution was
changed every 5 mln, to one containing various concentrations transport inhibitor in addition to the PGF2a.
of a PG
At the end of each concen-
tration series the inhibitor free solution was again perfused and aliquots were collected to test reversibility.
Allquots of the infusion fluid
and the effluent fractions were assayed for PGF2o by immunoassay
(Clinical
Assays, Inc., Cambridge, Mass.). In the absence of a PG transport inhibitor,
less than 5% of the
PGF2a present in the perfusion fluid was recovered in the effluent (Table i, Column A), indicating that at least 95% of the PGF2a was metabolized during a single passage through the lungs.
The antiserum
used in these experiments showed about 5% cross reaction with 15-ketoPGF2a (produced by direct oxidation of PGF2u; see ref. ii).
Since 15-
keto-PGF2u is the major product of pulmonary PGF2u metabolism,
a 5%
cross reaction with this product could result in an apparent 4.6% recovery of PGF2a even if the effluent contained no PGF2a.
Thus, the
4.6% value for the recovery of PGF2u must be an overestimation and may, in fact, represent an essentially complete metabolism of the infused PGF2u. When the perfusion fluid contained 3 mM of probenecid,
85% of the
PGF2u was recovered in the effluent, indicating an 84% inhibition of pulmonary PGF2a metabolism (Table I, Columns A and C).
(Probenecid,
benemld, was a gift from Merck, Sharp & Dohme Research Laboratories, West Point, Pa.).
This concentration of probenecid, which is the
concentration typically used to inhibit organic acid transport, seems to be required since 0.i mM had little or no inhibitory effect.
634
OCTOBER 1975
VOL. 10 NO. 4
PROSTAGLANDINS
Table i.
The effect of probenecld and bromcresol green on the recovery
of PGF2a infused through the isolated rat lung or incubated with the hlgh-speed supernatant fraction of homogenized rat lungs. *
" Percent Inhibition of PGF2a Metabolism*
Percent PGF2a Recovery (A) Perfused Lung
Inhibitor mM
4.6±0.3 (12)
None
Probenecid 0.1 3.0 Bromcresol green 0.001 0.01
(c)
(B)
(D)
Perfused Lung
Homogenized Lung
Homogenized Lung
23.2±0.2 (6)
6.2±1.3 (8) 84,8±3.9 (5)
83.1+-1.8 (6)
1.8±0.6 (8) 84.1±3.0 (5)
78.0±2.1 (8)
60.9±1.7 (7) 95.9±1.8 (5)
23.6±0.9 (5) 26.2±2.6 (6)
50.2±1.4 (7) 95.7±!.7 (5)
0.2±0.9 (5) 3.9±2,4 (6)
Mean ± o n e S . E .
(n)
Bromcresol green was found to be some 300-fold more potent than probenecid on this system, resulting in an essentially complete (96%) inhibition of PGF2u metabolism at a concentration of 0.01 mM (Table i, Column C).
Some inhibition was noted at a bromcresol green concentration
as low as 5 x 10-8M.
These effects of probenecld end bromcresol green
were reversed after 2-5 mln of perfuslon with inhibitor free solution. Although probenecid is known to be an organic acid transport inhibitor, the possibility that it also inhibits 15-hydroxy-prostaglandln dehydrogenase, and/or other enzymes of pulmonary PG metabolism, had to be tested.
Allquots of the hlgh-speed supernatant fraction of
homogenized rat lungs (12) were made up in Bucher medium containing 3HPGF2u (New England Nuclear Corporation, (13).
Boston, Mass.) and 2 m M N A D
Following i0 min incubation in the presence or absence of various
concentrations of probenecld or bromeresol green, extracts (14) of these supernatants were chromatographed, together with authentic PGF2a, on silica gel impregnated paper (AI system; see ref. 15).
In the presence
of 3 mM probenecld PGF2a metabolism by this cell-free supernatant fraction was inhibited by 78% (Table i, Column D).
This is comparable
to the inhibition caused by this probenecld concentration on PGF2a metabolism by the intact lung (Table i, Column C).
OCTOBER 1975 VOL. 10 NO. 4
We must, therefore,
635
PROSTAGLANDINS
consider the possibility that the probenecid-induced inhibition of PG metabolism
by the pe~fused lung was
due to the inhibition of enzymatic
activity rather than inhibition of transmembrane transport.
In the case
of bromcresol green we can, however, rule out a direct effect on the enzyme system since 0 . O l m M b r o m c r e s o l
green, which caused an essentially
maximal inhibition of PG metabolism by the perfused lung, did not have a significant inhibitory effect on PG metabolism by the cell-free supernatant (Table I, C o l u ~ D).
Thus the inhibitory effect of bromcresol
green on the PG metabolism by the Isolated-perfused lung must be associated with a system not present in the high-speed supernatant fraction of lung homogenate, namely cellular membranes. In order to test the concept that the pulmonary metabolism of PGs is preceded by a carrler-mediated transmembrane transport into an intracellular compartment(s), we have compared the clearance time of 3HPGF2u across the isolated rat lung to that of a purely extracellular substance, 14C-sucrose, in the absence and presence of PG transport inhibltors.
A mixture of 3H-PGF2u and 14C-sucrose (New England Nuclear
Corp.) was injected into the infusion cannula close to the pulmonary artery as a single bolus in 0.I ml of saline.
Aliquots of effluent were
collected at 1.5 to 3 sec intervals during the first minute and at i0 sec intervals for an additional minute. simultaneously for 3H and 14C.
These samples were counted
The counts per minute were corrected for
isotope spill-over and efficiency and the 3H and 14C disintegrations per min (DPM) were calculated. The highest 14C concentration was observed in the effluent at 5 to i0 sec after injection and 50% of the originally injected 14C was cleared through the lung in less than 6 sec after the initial appearance of the isotopes (Fig. i).
In the absence of a PG transport inhibitor the
occurrence of the peak 3H concentration in the effluent was greatly delayed as compared to the 14C peak (Fig. IA) taking over 24 sec to clear 50% of the 3H through the lung.
When the perfuslon medium contained
0.01 mM bromcresol green, the 14C curve remained unaffected (Fig. IB) while the 3H curve was shifted to the left showing a peak 3H clearance indistinguishable from the 14C peak and a 50% clearance time only slightly greater than the half-tlme of 14C clearance.
Similar results were
obtained with 3 mMprobenecid while fursemlde, an effective inhibitor of
636
OCTOBER 1975
VOL. 10 NO. 4
PROSTAGLANDINS
1207
A, CONTROL
80
I-!
~ B' BROMCRESOLGREEN'IO-SM
4C
I
I>
/
I t~
".J
olr t"
t I",,
""
'"
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Figure i:
20
........
/ I
Xo
INHIBITION OF PULMONARY PROSTAGLANDIN METABOLISM BY INHIBITORS OF PROSTAGLANDIN BIOTRANSPORT (PROBENECID AND BROMCRESOL GREEN).
L. Z. Bito and R. A. Baroody
Ophthalmology Research, Columbia University College of Physicians and Surgeons, New York, N.Y. 10032
The transfer of prostaglandins (PGs) across the blood-brain (I) and blood-aqueous barriers (2), and their absorption from the lumen of the rabbit vagina (3) is mediated by saturable, "carrier-mediated", transport processes.
In vitro studies demonstrated that such a PG transport
process can take place against a concentration gradient (4) and thus it may, under certain conditions, represent an active transport process. The physiological significance of PG transport processes has been underscored by the observation that rabbit red blood cells, and thus presumably the basic cell membrane in general, are impermeable to PGs (5,6).
These observations indicate that the flux of PGs across some
specialized membranes is mediated by special mechanisms rather than being accounted for by simple diffusion. An earlier survey of concentrative PG accumulation by animal tissues (7,8) suggested that in addition to the chorold plexus, ciliary processes and kidney cortex, the mammalian lung also possesses such PG transport mechanisms.
Since the key enzymes in pulmonary PG metabolism
must be intracellular, the effective metabolism of PGs during their passage through the lungs must require an initial step of carriermediated transmembrane transport (7,9). The extent of 3H accumulation by the rat lungs, following shortterm intravenous infusion of 3H-PGF2a, is drastically reduced in animals which were pretreated with a PG transport inhibitor (i0). Experiments currently in progress show that two PG transport inhibitors, probenecid and bromcresol green, both inhibit the metabolism of PGF2a by the isolated perfused rat lung.
One of these substances, namely bromcresol green,
has no inhibitory effect on the metabolism of this PG by the highspeed supernatant fraction of lung homogenates.
Io-15-75
PROSTAGLANDINS OCTOBER 1975
VOL. 10 NO. 4
633
PROSTAGLANDINS
Columbla-Sherman Chloropent
albino rats (400-450 gm) were anesthetized with
(Fort Dodge Laboratories,
Inc., Fort Dodge, Iowa).
The heart
was exposed and heparln (500 units), in saline, was injected into the right ventricle.
The heart and lungs were removed and a cannula was
inserted into the pulmonary artery.
This preparation,
suspended in a
moist chamber maintained at 37°C, was infused at a rate of 8 ml/min with Kreb's solution gassed with 95% 02 and 5% CO 2.
After 15 min the in-
fusion solution was changed to one containing I00 ng/ml of PGF2~ and three effluent samples were collected at I mln intervals.
The PGF2a was
kindly supplied by Dr. John E. Pike of The Upjohn Company (Kalamazoo, Mich.).
After this initial control perfusion the infusion solution was
changed every 5 mln, to one containing various concentrations transport inhibitor in addition to the PGF2a.
of a PG
At the end of each concen-
tration series the inhibitor free solution was again perfused and aliquots were collected to test reversibility.
Allquots of the infusion fluid
and the effluent fractions were assayed for PGF2o by immunoassay
(Clinical
Assays, Inc., Cambridge, Mass.). In the absence of a PG transport inhibitor,
less than 5% of the
PGF2a present in the perfusion fluid was recovered in the effluent (Table i, Column A), indicating that at least 95% of the PGF2a was metabolized during a single passage through the lungs.
The antiserum
used in these experiments showed about 5% cross reaction with 15-ketoPGF2a (produced by direct oxidation of PGF2u; see ref. ii).
Since 15-
keto-PGF2u is the major product of pulmonary PGF2u metabolism,
a 5%
cross reaction with this product could result in an apparent 4.6% recovery of PGF2a even if the effluent contained no PGF2a.
Thus, the
4.6% value for the recovery of PGF2u must be an overestimation and may, in fact, represent an essentially complete metabolism of the infused PGF2u. When the perfusion fluid contained 3 mM of probenecid,
85% of the
PGF2u was recovered in the effluent, indicating an 84% inhibition of pulmonary PGF2a metabolism (Table I, Columns A and C).
(Probenecid,
benemld, was a gift from Merck, Sharp & Dohme Research Laboratories, West Point, Pa.).
This concentration of probenecid, which is the
concentration typically used to inhibit organic acid transport, seems to be required since 0.i mM had little or no inhibitory effect.
634
OCTOBER 1975
VOL. 10 NO. 4
PROSTAGLANDINS
Table i.
The effect of probenecld and bromcresol green on the recovery
of PGF2a infused through the isolated rat lung or incubated with the hlgh-speed supernatant fraction of homogenized rat lungs. *
" Percent Inhibition of PGF2a Metabolism*
Percent PGF2a Recovery (A) Perfused Lung
Inhibitor mM
4.6±0.3 (12)
None
Probenecid 0.1 3.0 Bromcresol green 0.001 0.01
(c)
(B)
(D)
Perfused Lung
Homogenized Lung
Homogenized Lung
23.2±0.2 (6)
6.2±1.3 (8) 84,8±3.9 (5)
83.1+-1.8 (6)
1.8±0.6 (8) 84.1±3.0 (5)
78.0±2.1 (8)
60.9±1.7 (7) 95.9±1.8 (5)
23.6±0.9 (5) 26.2±2.6 (6)
50.2±1.4 (7) 95.7±!.7 (5)
0.2±0.9 (5) 3.9±2,4 (6)
Mean ± o n e S . E .
(n)
Bromcresol green was found to be some 300-fold more potent than probenecid on this system, resulting in an essentially complete (96%) inhibition of PGF2u metabolism at a concentration of 0.01 mM (Table i, Column C).
Some inhibition was noted at a bromcresol green concentration
as low as 5 x 10-8M.
These effects of probenecld end bromcresol green
were reversed after 2-5 mln of perfuslon with inhibitor free solution. Although probenecid is known to be an organic acid transport inhibitor, the possibility that it also inhibits 15-hydroxy-prostaglandln dehydrogenase, and/or other enzymes of pulmonary PG metabolism, had to be tested.
Allquots of the hlgh-speed supernatant fraction of
homogenized rat lungs (12) were made up in Bucher medium containing 3HPGF2u (New England Nuclear Corporation, (13).
Boston, Mass.) and 2 m M N A D
Following i0 min incubation in the presence or absence of various
concentrations of probenecld or bromeresol green, extracts (14) of these supernatants were chromatographed, together with authentic PGF2a, on silica gel impregnated paper (AI system; see ref. 15).
In the presence
of 3 mM probenecld PGF2a metabolism by this cell-free supernatant fraction was inhibited by 78% (Table i, Column D).
This is comparable
to the inhibition caused by this probenecld concentration on PGF2a metabolism by the intact lung (Table i, Column C).
OCTOBER 1975 VOL. 10 NO. 4
We must, therefore,
635
PROSTAGLANDINS
consider the possibility that the probenecid-induced inhibition of PG metabolism
by the pe~fused lung was
due to the inhibition of enzymatic
activity rather than inhibition of transmembrane transport.
In the case
of bromcresol green we can, however, rule out a direct effect on the enzyme system since 0 . O l m M b r o m c r e s o l
green, which caused an essentially
maximal inhibition of PG metabolism by the perfused lung, did not have a significant inhibitory effect on PG metabolism by the cell-free supernatant (Table I, C o l u ~ D).
Thus the inhibitory effect of bromcresol
green on the PG metabolism by the Isolated-perfused lung must be associated with a system not present in the high-speed supernatant fraction of lung homogenate, namely cellular membranes. In order to test the concept that the pulmonary metabolism of PGs is preceded by a carrler-mediated transmembrane transport into an intracellular compartment(s), we have compared the clearance time of 3HPGF2u across the isolated rat lung to that of a purely extracellular substance, 14C-sucrose, in the absence and presence of PG transport inhibltors.
A mixture of 3H-PGF2u and 14C-sucrose (New England Nuclear
Corp.) was injected into the infusion cannula close to the pulmonary artery as a single bolus in 0.I ml of saline.
Aliquots of effluent were
collected at 1.5 to 3 sec intervals during the first minute and at i0 sec intervals for an additional minute. simultaneously for 3H and 14C.
These samples were counted
The counts per minute were corrected for
isotope spill-over and efficiency and the 3H and 14C disintegrations per min (DPM) were calculated. The highest 14C concentration was observed in the effluent at 5 to i0 sec after injection and 50% of the originally injected 14C was cleared through the lung in less than 6 sec after the initial appearance of the isotopes (Fig. i).
In the absence of a PG transport inhibitor the
occurrence of the peak 3H concentration in the effluent was greatly delayed as compared to the 14C peak (Fig. IA) taking over 24 sec to clear 50% of the 3H through the lung.
When the perfuslon medium contained
0.01 mM bromcresol green, the 14C curve remained unaffected (Fig. IB) while the 3H curve was shifted to the left showing a peak 3H clearance indistinguishable from the 14C peak and a 50% clearance time only slightly greater than the half-tlme of 14C clearance.
Similar results were
obtained with 3 mMprobenecid while fursemlde, an effective inhibitor of
636
OCTOBER 1975
VOL. 10 NO. 4
PROSTAGLANDINS
1207
A, CONTROL
80
I-!
~ B' BROMCRESOLGREEN'IO-SM
4C
I
I>
/
I t~
".J
olr t"
t I",,
""
'"
]0
Figure i:
20
........
/ I
Xo
