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Tranylcypromine is not a selective inhibitor of prostacyclin in rats
THROMBOSIS @ Pergamon
RESEARCH 14; 245-248 Press Ltd.1979. Printed
in Great
Britain
oo4+~848/~~/olol-o24~
Jb02.00/0
COMMUNICATION
BRIEF
TRANYLCYPROMINE IS NOT A SELECTIVE INHIBITOR OF PROSTACYCLIN IN RATS Grazyna Rajtar' and Giovanni de Gaetano Laboratory of Cardiovascular Clinical Pharmacology Istituto di Ricerche Farmacologiche "Mario Negri" Via Eritrea, 62 - 20157 MILAN, Italy (Received
21.9.1978; Accepted by
in revised form 13.10.1978. Editor A.L. Bloom)
INTRODUCTION Recent developments in prostaglandin (PG) metabolism indicate that cyclic endoperoxides (PGG2 and PGH2) may undergo different enzymatic transformations in the presence of microsomes from different sources; indeed, microsomes from pig aorta ormesenteric
arteries, rabbit aorta and rat stomach fundus meta-
bolize PG endoperoxides to PG12, whereas microsomes from rat stomach corpus or liver, rabbit lungs, spleen brain or kidney medulla and from ram seminal vescicles transform PG endoperoxides to thromboxane A2 (TXA2) and stable PGs (1). Intact platelets and dissected vascular specimens are also able to metabolize both arachidonic acid and PG endoperoxides to TXA2 and PG12, respectively (2,3).
Since PG12 and TXA2 show opposite effects on platelet aggre-
gation and vascular tone (3), it is important to find pharmacological means to specifically inhibit either metabolic pathway.
Gryglewski et al. (1)
reported that tranylcypromine effectively inhibited (IC50 : 160 I_'g/ml)PG12 formation from PG endoperoxides by pig aorta microsomes, but was completely ineffective (at concentration of 1 mg/ml) on TXA2 generation by horse platelet microsomes.
They concluded that this mono-amine oxidase inhibitor
selectively inhibits PG12 synthesis.
Tranylcypromine has been subsequently
used by several investigators (see Ref.4 ) as a pharmacological mean to +Visiting Scientist, on leave of absence from the Department of Pharmacology, Medical Academy, Lublin, Poland. 245
246
TFUNYLCYPROMINE
AM) PROSTACYCLIN
Vol.14,No.l
characterize PG12 formation in various experimental systems. Bourgain (4) has also suggested that selective inhibition of PG12 synthesis could explain the observed enhancement
by tranylcypromine (345-690 pg/ml) superfusion of ex-
perimental platelet thrombosis in exposed branches of the mesenteric artery in Wistar rats. During the course of a study on the effect of different drugs on platelet and vascular PG synthesis in the rat, we have investigated tranylcypromine. Malondialdehyde (MDA) formation synthesis (5) -
an indicator of platelet prostaglandin
was determined by measuring the amount of MDA generated in
rat platelet rich plasma upon stimulation with thrombin (6).
PG12 activity released from rat abdominal aorta and inferior vena cava,was measured as platelet aggregation inhibiting potency in an Elvi 840 aggregometer as previously described (6).
Male CD rats (280-350 gr) from Charles
River Italia were used in this study. Tranylcypromine sulphate was dissolved in water or in the buffers used for
PG12 activity determination.
In vitro experiments showed that preincubation
of this drug (for 1 min at 37Y)
with rat platelet-rich plasma (about 1.4~10'
platelets/ml) resulted in a concentration dependent inhibition of thrombinstimulated MDA formation (Table).
The calculated IC50 was 87 pg/ml, a value
in good agreement with the IC50 reported by Griglewski et al. (1) on pig aortic microsomes (160 pg/ml).
When tranylcypromine was preincubated with
vascular specimens in 0.05M 'Tris' buffer, pH 9.2, for up to 60 min, the release of PG12 activity was also inhibited (Table).
The calculated IC50
values were 42 yg/ml and 470 I_'g/mlfor venous and arterial vessels,respectively.
Experiments (not reported in the Table) using 0.05M 'Tris' buffer
pH 7.4, and 5 min preincubation gave similar results.
In preliminary exper-
iments we were unable to find any consistent inhibitory effect on either platelet MDA.or vascular PG12 activity generated in vitro following i.p. or i.v. injections of tranylcypromine (single doses ranging between 5 and 60 mg/kg b.w. or daily doses of 5 mg/kg b.w. for 6 days).
Rats were sacrificed
between 5 and 12 hours after the last drug administration. The ID50 of tranylcypromine as an inhibitor of mono-amine oxidases was around 6 mg/kg b.w. (16 hours after a single i.p. administration). Our data indicate that using intact platelets and vascular specimens from the same animal species (the rat) rather than microsomal preparations from different species, a selective inhibitory activity of tranylcypromine on vascular PG12 synthesis could not be found.
Indeed, in the in vitro
systems
used, platelets appeared to be as sensitive to tranylcypromine as veins which
Vol.14,No.l
TRANYLCYPROMINE
AND PROSTACYCLIN
247
TABLE In vitro effect of tranylcypromine on rat platelet MDA and vascular PGI2 generatlbn + . Mean (t S.E.) percent inhibition of these activities in respect to appropriate-controls is reported. Each value represents the mean of 5 to 12 experiments. TRANYLCYPROMINE ()rg/ml)
PLATELET MDA
VASCULAR PGI,-LIKE ACTIVITY VENOUS
I
ARTERIAL
I 2,000 1,000 750 500 300 200 100 50 10 5
89.16 + 3.12
81,33 + 6.47
83.52 + 3.84
79.16 t 7.95
67.12 + 2.90 35.96 T 8.51 25.24 z10.40
82.33 + 41.00 T 27.00 T 22.00 T <3
1
4.20 5.19 5.00 5.00
81.57 68.47 52.83 45.21 44.33 33.50 5.66
t 6.78 T 5.42 T16.22 T 9.50 T19.9 a4.98 z 2.40
' Platelet MDA formation was stimulated by thrombin (25 MIH u/ml) and measured following a tiobarbiturate reaction -
by spectrophotometric
readings at 532 nm (Smith et al., (5) ; Livio et al., (6) ). Vascular PG12like activi,tywas measured as platelet aggregation inhibitory potency of aliquots of supernatant buffer in which dissected vascular specimens (5 mg wet weight) had been incubated at room temperature.
The anti-
aggregating activity released from vessels was characterized by a number of criteria indicative of it being PG12 (instability and inactivation at high temperature and at acidic pH, inhibition by addition to the buffer of cycle-oxygenase inhibitors (aspirin, indomethacin), potentiation by phosphodiesterase inhibitors (theophylline,dipyridamole), activity on human and rat platelets).
_______________ were more sensitive than arteries.
It seems therefore reasonable to cast
some doubts about a selective mechanism of action of tranylcyprcnnine and to avoid extrapolation from one experimental system to another, concerning the relative efficacy of this drug on the complex metabolic pathways of arachidonic acid.
248
TRANYLCYPROMINE
AND PROSTACYCLIN
Vol.14,No.l
Failure to detect any ex vivo effect of tranylcypromine following its parenteral administration suggests that the potential clinical relevance of the effects observed in vitro
may be scarce. ACKNOWLDEGEMENTS
We thank Prof. M.J. SILVER (Cardeza Foundation,Thomas Jefferson University, Philadelphia, Pa., USA, Johananoff Fellow 1977-78 at the Mario Negri Institute) for helpful discussion.
Judith BAGGOTT, Gigliola BRAMBILLA and Paola
SEMINAR1 helped in preparing the manuscript. REFERENCES 1.
GRYGLEWSKI, R.J., BUNTING, S., MONCADA, S., FLOWER, R.J. and VANE, J.R.: Arterial walls are protected against deposition of platelet thrombi by a substance (Prostaglandin-X)which they make from prostaglandin endoperoxides. Prostaglandins -12, 685, 1976.
2.
BILLS, T.K., SMITH, J.B. and SILVER, M.J.: Metabolism of L14C7arachidonic acid by human platelets. Biochim.Biophys.Acta 424, 303, 197%.
3.
Prostacyclin platelet aggregation and MONCADA, S. and VANE, J.R.: thrombosis. In: Platelets: A Multidisciplinary Approach. Eds. de Gaetano, G. and Garattini, S. 1978, Raven Press, New York, p. 239.
4.
BOURGAIN, R.H.: Inhibition of PGI (Prostacyclin) synthesis in the arterial wall enhances the format ?on of white platelet thrombi in vivo. Haemostasis 1, 252, 1978.
5.
SMITH, J.B., INGERMAN, C.M. and SILVER, M.J,: Malondialdehyde formation as an indicator of prostaglandin production by human platelets. J. Lab. Clin. Med. 8& 167, 1976.
6.
LIVIO, M., VILLA, S. and de GAETANO, G.: Aspirin, thromboxane, and prostacyclin in rat: A dilemma resolved? -Lancet I, 1307,, 1978.
RESEARCH 14; 245-248 Press Ltd.1979. Printed
in Great
Britain
oo4+~848/~~/olol-o24~
Jb02.00/0
COMMUNICATION
BRIEF
TRANYLCYPROMINE IS NOT A SELECTIVE INHIBITOR OF PROSTACYCLIN IN RATS Grazyna Rajtar' and Giovanni de Gaetano Laboratory of Cardiovascular Clinical Pharmacology Istituto di Ricerche Farmacologiche "Mario Negri" Via Eritrea, 62 - 20157 MILAN, Italy (Received
21.9.1978; Accepted by
in revised form 13.10.1978. Editor A.L. Bloom)
INTRODUCTION Recent developments in prostaglandin (PG) metabolism indicate that cyclic endoperoxides (PGG2 and PGH2) may undergo different enzymatic transformations in the presence of microsomes from different sources; indeed, microsomes from pig aorta ormesenteric
arteries, rabbit aorta and rat stomach fundus meta-
bolize PG endoperoxides to PG12, whereas microsomes from rat stomach corpus or liver, rabbit lungs, spleen brain or kidney medulla and from ram seminal vescicles transform PG endoperoxides to thromboxane A2 (TXA2) and stable PGs (1). Intact platelets and dissected vascular specimens are also able to metabolize both arachidonic acid and PG endoperoxides to TXA2 and PG12, respectively (2,3).
Since PG12 and TXA2 show opposite effects on platelet aggre-
gation and vascular tone (3), it is important to find pharmacological means to specifically inhibit either metabolic pathway.
Gryglewski et al. (1)
reported that tranylcypromine effectively inhibited (IC50 : 160 I_'g/ml)PG12 formation from PG endoperoxides by pig aorta microsomes, but was completely ineffective (at concentration of 1 mg/ml) on TXA2 generation by horse platelet microsomes.
They concluded that this mono-amine oxidase inhibitor
selectively inhibits PG12 synthesis.
Tranylcypromine has been subsequently
used by several investigators (see Ref.4 ) as a pharmacological mean to +Visiting Scientist, on leave of absence from the Department of Pharmacology, Medical Academy, Lublin, Poland. 245
246
TFUNYLCYPROMINE
AM) PROSTACYCLIN
Vol.14,No.l
characterize PG12 formation in various experimental systems. Bourgain (4) has also suggested that selective inhibition of PG12 synthesis could explain the observed enhancement
by tranylcypromine (345-690 pg/ml) superfusion of ex-
perimental platelet thrombosis in exposed branches of the mesenteric artery in Wistar rats. During the course of a study on the effect of different drugs on platelet and vascular PG synthesis in the rat, we have investigated tranylcypromine. Malondialdehyde (MDA) formation synthesis (5) -
an indicator of platelet prostaglandin
was determined by measuring the amount of MDA generated in
rat platelet rich plasma upon stimulation with thrombin (6).
PG12 activity released from rat abdominal aorta and inferior vena cava,was measured as platelet aggregation inhibiting potency in an Elvi 840 aggregometer as previously described (6).
Male CD rats (280-350 gr) from Charles
River Italia were used in this study. Tranylcypromine sulphate was dissolved in water or in the buffers used for
PG12 activity determination.
In vitro experiments showed that preincubation
of this drug (for 1 min at 37Y)
with rat platelet-rich plasma (about 1.4~10'
platelets/ml) resulted in a concentration dependent inhibition of thrombinstimulated MDA formation (Table).
The calculated IC50 was 87 pg/ml, a value
in good agreement with the IC50 reported by Griglewski et al. (1) on pig aortic microsomes (160 pg/ml).
When tranylcypromine was preincubated with
vascular specimens in 0.05M 'Tris' buffer, pH 9.2, for up to 60 min, the release of PG12 activity was also inhibited (Table).
The calculated IC50
values were 42 yg/ml and 470 I_'g/mlfor venous and arterial vessels,respectively.
Experiments (not reported in the Table) using 0.05M 'Tris' buffer
pH 7.4, and 5 min preincubation gave similar results.
In preliminary exper-
iments we were unable to find any consistent inhibitory effect on either platelet MDA.or vascular PG12 activity generated in vitro following i.p. or i.v. injections of tranylcypromine (single doses ranging between 5 and 60 mg/kg b.w. or daily doses of 5 mg/kg b.w. for 6 days).
Rats were sacrificed
between 5 and 12 hours after the last drug administration. The ID50 of tranylcypromine as an inhibitor of mono-amine oxidases was around 6 mg/kg b.w. (16 hours after a single i.p. administration). Our data indicate that using intact platelets and vascular specimens from the same animal species (the rat) rather than microsomal preparations from different species, a selective inhibitory activity of tranylcypromine on vascular PG12 synthesis could not be found.
Indeed, in the in vitro
systems
used, platelets appeared to be as sensitive to tranylcypromine as veins which
Vol.14,No.l
TRANYLCYPROMINE
AND PROSTACYCLIN
247
TABLE In vitro effect of tranylcypromine on rat platelet MDA and vascular PGI2 generatlbn + . Mean (t S.E.) percent inhibition of these activities in respect to appropriate-controls is reported. Each value represents the mean of 5 to 12 experiments. TRANYLCYPROMINE ()rg/ml)
PLATELET MDA
VASCULAR PGI,-LIKE ACTIVITY VENOUS
I
ARTERIAL
I 2,000 1,000 750 500 300 200 100 50 10 5
89.16 + 3.12
81,33 + 6.47
83.52 + 3.84
79.16 t 7.95
67.12 + 2.90 35.96 T 8.51 25.24 z10.40
82.33 + 41.00 T 27.00 T 22.00 T <3
1
4.20 5.19 5.00 5.00
81.57 68.47 52.83 45.21 44.33 33.50 5.66
t 6.78 T 5.42 T16.22 T 9.50 T19.9 a4.98 z 2.40
' Platelet MDA formation was stimulated by thrombin (25 MIH u/ml) and measured following a tiobarbiturate reaction -
by spectrophotometric
readings at 532 nm (Smith et al., (5) ; Livio et al., (6) ). Vascular PG12like activi,tywas measured as platelet aggregation inhibitory potency of aliquots of supernatant buffer in which dissected vascular specimens (5 mg wet weight) had been incubated at room temperature.
The anti-
aggregating activity released from vessels was characterized by a number of criteria indicative of it being PG12 (instability and inactivation at high temperature and at acidic pH, inhibition by addition to the buffer of cycle-oxygenase inhibitors (aspirin, indomethacin), potentiation by phosphodiesterase inhibitors (theophylline,dipyridamole), activity on human and rat platelets).
_______________ were more sensitive than arteries.
It seems therefore reasonable to cast
some doubts about a selective mechanism of action of tranylcyprcnnine and to avoid extrapolation from one experimental system to another, concerning the relative efficacy of this drug on the complex metabolic pathways of arachidonic acid.
248
TRANYLCYPROMINE
AND PROSTACYCLIN
Vol.14,No.l
Failure to detect any ex vivo effect of tranylcypromine following its parenteral administration suggests that the potential clinical relevance of the effects observed in vitro
may be scarce. ACKNOWLDEGEMENTS
We thank Prof. M.J. SILVER (Cardeza Foundation,Thomas Jefferson University, Philadelphia, Pa., USA, Johananoff Fellow 1977-78 at the Mario Negri Institute) for helpful discussion.
Judith BAGGOTT, Gigliola BRAMBILLA and Paola
SEMINAR1 helped in preparing the manuscript. REFERENCES 1.
GRYGLEWSKI, R.J., BUNTING, S., MONCADA, S., FLOWER, R.J. and VANE, J.R.: Arterial walls are protected against deposition of platelet thrombi by a substance (Prostaglandin-X)which they make from prostaglandin endoperoxides. Prostaglandins -12, 685, 1976.
2.
BILLS, T.K., SMITH, J.B. and SILVER, M.J.: Metabolism of L14C7arachidonic acid by human platelets. Biochim.Biophys.Acta 424, 303, 197%.
3.
Prostacyclin platelet aggregation and MONCADA, S. and VANE, J.R.: thrombosis. In: Platelets: A Multidisciplinary Approach. Eds. de Gaetano, G. and Garattini, S. 1978, Raven Press, New York, p. 239.
4.
BOURGAIN, R.H.: Inhibition of PGI (Prostacyclin) synthesis in the arterial wall enhances the format ?on of white platelet thrombi in vivo. Haemostasis 1, 252, 1978.
5.
SMITH, J.B., INGERMAN, C.M. and SILVER, M.J,: Malondialdehyde formation as an indicator of prostaglandin production by human platelets. J. Lab. Clin. Med. 8& 167, 1976.
6.
LIVIO, M., VILLA, S. and de GAETANO, G.: Aspirin, thromboxane, and prostacyclin in rat: A dilemma resolved? -Lancet I, 1307,, 1978.