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Human plasma transforms prostacyclin (PGI2) into a platelet antiaggregatory substance which contracts isolated bovine coronary arteries
PROSTAGLANDINS HUMAN PLASMA TRANSFORMS PROSTACYCLIN A PLATELET ANTIAGGREGATORY SUBSTANCE ISOLATED BOVINE CORONARY ARTERIES(l).
(PG12) INTO WHICH
CONTRACTS
+* *+ M.F.Glmeno+, L.Sterin-Borde , E.S.Borda+*, M.A.Lazzarl and A.L.Gimeno* Centro de Estudios Farmacolbglcos y de Principios Naturales (CEFAPRIN), CONICET, V. de Obligado 2490, 1428 Buenos Aires, ARGENTINA and Instituto de Investigaciones Hemato16gicas. Academia National de Mediclna. Pscheco de Melo 3081, 1425 Buenos Aires, ARGENTINA. ABSTRACT Prostacyclin (PG12) incubated In Human Platelet Rich Flasma (PAP); in Platelet Poor Plasma (PPP) or in Hrebs-RingerBicarbonate (HRB) during different periods of time on contractions of bovine coronary arteries and on the ADP platelet aggnzgative capacity of human PAP, were explored. It was documented that incubates In PRP or in PPP retain an antiaggregatory activity at higher levels and during a longer time than in HR6.h the other hand, PGI2 Incubates In KRB exhibited only a relaxing activity on Isolated bovine coronary arteries, whereas when incubated In PRP or in PPP presented a biphasic influence. 'The initial effects (evoked by incubates of 30 minutes) were distinctly relaxing but those obtained with later incubates (60150 minutes) stimulated clearly the resting basal tone of the arteries. The possibility that the human plasma might have an enzyme(s) able to transform prostacyclin into a more stable material with human antiaggregatory platelet function and bovine coronary Mailing
contracting Address:
capacity
is
A.L. GIMENCJ,M.D. V. de Gbligado 2490 1428 Buenos Aires, ARGENTINA
(1) This work has been supported National
discussed.
de Investigaclones
by Grant Clentiflcas
6630 from the Consejo y T&cnlcas de la
Repcbllca Argentina (CONICCT). * Senior Investigator, ** Junior
Investigator,
CONICET. CONICET.
INTRODUCTIUN Prostscyclin (FIG1 > is en unstable prostaglandin having a particularly short haff life. The menouremcnt of its rate of release from a tissue nnt only depends on the rate of production and diffusion out of the tissue but also on the ability
JUNE
1980 VOL. 19 NO. 6
907
PROSTAGLANDINS of the tissue and the incubation media to degrade it. We have recently shown that a PGI2-like material produced by the rat stomach fundus retains its antiaggregatory activity in human plasma or serum during a period of time significantly longer than in Hrebs solution (I). The fact that one of the biological activities of a PGI2-like substance persist in plasma or serum more than expected from stability studies of PGI2 in aqueous solutions (2,3) made us to suggest the possibility that the unstable prostaglandin material might be transformed into another compound also having antiaggregatory activity. The present experiments were designed in order to a8certain the validity of this hypothesis. Authentic PGI2 instead of PGI2-like material synthetized by rat stomach Pundus was used in order to avoid the possible role of other substances which might be present in the incubation medium. Prostacyclin was incubated in human platelet-rich plasma (PRP), human plateletpoor plasma (PPP) and Hrehs-Ringer-Bicarbonate (KRS) during different periods of time. The biological activity of the incubates were tested on contractions of isolated bovine coronary arteries and on the ADP induced platelet aggregation of human platelet-rich plasma. MATERIALAND METHODS (I)
The following materials were used: (a) Prostacyclin sodium salt (*Iwas dissolved in 0.05 M Tris buffer (pH 9) on the day of the experiment; 6-k&o-PGEq(*) was first dissolved in ethanol and the day to be used in aliquots of this solution were diluted with Hrebs solution and bketo-PGF&(*) was prepared directly in KREI. (b) PGI2 and &keto-PGFTC( were additioned to: KrebsRinger-Bicarbonate composed as reported elsewhere (4); to human platelet-rich plasma prepared by centrifuging the blood for 10 min at 200 g and to human platelet-poor plasma obtained by centrifuging the blood after the PRP had been removed at 15Ofl g for 30 min. The blood was obtained from donors which had not taken aspirin for at least 7 days and collected into tubes containing Na citrate 3.1% (9:l v.v.). PGI was also added to KRS containing human serum albumin (4 and 6%). The pH of HHH, PKP and PPP determined at room temperature was 0.0 * 0.1. The following test were performed: (a> Antiaggregatory sctivity. Prostacyclin in Tris buffer solution (16 ng in 16 ,ul) were added to 3 test tubes containing 400 ~1 of KHS, PRP and PPP respectively. Aliquots of 50 ul (with 2 ng of PGI2)from each different media were tested for antiaggregatory activity using 400 ul of PUP in the aggregometer cuvette. This dose of PGI2 added to the PRP inhibited ADP-induced platelet aggregatim by approximately 90%. The aggregation was triggered by ADP at 5 JJMfinal concentration. Control measurements were performed (II)
908
JUNE 1980 VOL. 19 NO. 6
PROSTAGLANDINS after adminietratinn of 50 ul of KRS, PAP or PPP without prostacyclin. Light transmission through the sample was recorded as a measurement of aggregation. The highest recorded value of light transmission following ADP addition was taken as the maximal ADP aggregation (100%) for that bath of PRP. Forthwith, the 3 media plus PGIz were incubated in a thermostatized bath at 37 oC and samples of 50 ul were taken at 30, 60, 90 and 120 min. The antiaggregatory activity of PG12 in the various media at different incubation times was expressed as per cent of maximal aggregation induced by ADP in control condition. b) Tonic changes of isolated bovine coronary arteries. Bovine hearts of freshly slaughtered animals were immersed in cold KRS and transported to the laboratory. The left descending anterior coronary artery and several of its primary branches were dissected out separated free of fat and cut hellically. Arterial strips were mounted in 6 different baths (numerated from 1 to 6) containing 20 ml of KRS mixed with 5% CO2 in oxygen at 37 QC and pH 7.4. The tissues were allowed to equilibrate for at least 90 min under a resting tension of 2 g. The mechanical changes of the preparation elicited by experimental additions were expressed as mg of tension above or under this reeting or basal tension (Cl tension in the figures). A force transducer (Statham Gold-Cell-U C 31 coupled to an ink writing oscilloqraph (San EI IS0 System) were used. At 0 time following equilibration 0.5 ug of PGI2 (in 0.1 ml of KRG, PRP or PPP) were additioned toleach tissue bath giving a final . After its effect was recorded the concentration of 25 ng.ml preparations were washed 3 times and allowed to recover their basal level at least for 15 minutes. PGIz incubated in KRB was inactivated at 37 W and aliquots of 0.1 ml were taken at 15, 30, GO, 90, 120 and 150 min. The samples corresponding to 15,6U and 120 min were tested on the coronary strip mounted in the bath number 1, whereas those of 30, 90 and 150 min in the strip mounted in the bath number 2. The same procedure was followed for PRP + PGI2 and PPP + PGIz using the arterial strips mounted in baths number 3, 4 and 5, 6 respectively. This procedure allowed enough time for the complete recovery of the basal tone after washings. Identical experiments were also performed employing G-keto-PGF# incubated during different periods in PPP. Furthermore in some preparations several concentrations of non-incubated 6keto-PGL 1 kjere tested. Experimental results were compared employing the Differences between means uere considered sigStudent t test. nificant-if
(*>
JUNE
p = 0.05
or
less.
Kindly provided by Dr. J.E.Pike; Kalamazon, Michigan, U.S.A.
1980 VOL. 19 NO. 6
The Upjnhn
Company,
909
PROSTAGLANDINS
RESULTS
results nf the inactivating PG12 at 37 QC in different media are summarized in figure 1. It can be seen that almost all the antiaggregatory activity of PGI2 disappeared after 60 min when diluted in KRB. On the other hand, more than 5fl%of this biological action was still present when PG12 was during the ssme period of time incubated in PRP or PPP. The most surprising finding was that after 120 min of incubation the antiaggregatory effect of PGI only diminished around 5U% both in PRP or PPP. Inasmuch as p1asma proteins could alter the chemical hydrolysis of PG12 experiments were done adding prostacyclin to KRB containing human serum albumin. It was noticed that albumin failed to influence the inactivation time of PG12. In order to search whether in the presence of plasma PGI2 was transformed into another material with biological activity on isnlated vessels, prostacyclin was incubated in different media (HUE, PAP or PPP) at 37 QC and then tested over isolated bovine coronary arteries (Figure 2, panel (a) show that in HHB the typical relaxing action of P&I2 decreased as the incubation time progresses disappearing at 60 min when the unstable prost+ glandin becames inactivated. Following this time the arterial strips did not respond with any mechanical change to the additions. It is interesting to point out that the same result was obtained measuring the antiaggregatory activity of PG12 incubated in KRD solution. On the other hand, when PG12 was kept in PRP or PPP the characteristic initial inhibitory action of PG12 was also seen, diminishing with time up to 30 min.However starting from the 60 min trial the tension of the bovine coronary artery commence to increase, reaching the maximal response with incubates of 150 min (figure 2 panels (b) and cc>. After this period of time the increment in tone induced by PGI2 incubated in PAP or PPP was sensible minor (results not shown in figures). Controls with human plasma without prostacyclin run at Cl time and after 120 min of incubation at 37 QC failed to induce mechanical responses. Figure 3 depicts tracings showing the changes in tension of coronary arterial strips recorded when PG12 or 6-keto-PGFld were incubated during different times and then delivered to the tissue bath. The arteries presented a lack of responsiveness to the addition of 6-keto-PGFlclin PPP up to 30 min, but after 120 min of incubation the tissue reacts with a clear increment of the basal tone (figure 3, middle tracing). The magnitud of contractions became similar to that evoked by PG12 incubated during equivalent periods in PPP or to that obtained with 15 to 25 ng of 6-ketn-PGE1 (figure 3, upper and lower tracings, respectively). The
DISCUSSION We have recently demonstrated that a PGI?-like material synthetized by the isolated rat stomach fundus conserved during a
910
JUNE 1980 VOL. 19 NO. 6
PROSTAGLANDINS
\ 9
30
Figure
1.
4
16)
60 TIME (min)
I
90
1
120
Inhibition of ADP platelet eggregation (meeaured a8 the lnverae of % light transmission) evoked by PGI2 (2 ng) Incubated during different perioda of time in Krebs-Ringer-Bicarbonate (KRB); Human Platelet-Rich Plaama@RP) or Human Platelet-Poor Pleama (PPP). Points and vertical bare = Means * SEM. Figure6 between parentheaea = number of experiments.
JUNE 1980 VOL. 19 NO. 6
911
PROSTAGLANDINS
+100 +
50
r
c
I I I
I I
PGI2+ KRB
(n-9)
- 100
G
l
100
+
50
E
PG12+ PRP (n=7)
0
E 3;
5
I-
-
50
t
-100 +lOO
+
50
PGI,+ PPP (n=8)
0
-
50
- 100
Figure
912
2.
0 15 30 60 90 120 150 INCUBATION TIME (min 1
Resting tone (in mg) of ieolated bovine coronary arteries a8 influenced by PGI (25 ng.ml’1) incubated during different perioda oi: time in HRB, PRP or PPP. Tension prior PG12 taken a8 reetlng basal 0 levele. Columns and vertical bare- Meana * SEM. For other detaila aee text and Fig. 1.
JUNE
1980 VOL. 19 NO. 6
PROSTAGLANDINS
JUNE 1980 VOL. 19 NO. 6
913
PROSTAGLANDINS
longer time its platelet antiaggregatory capacity when incubated in human plasma or sera than when kept in HRE. In the present study, employing authentic prostacyclin, it was documented that no less than 50% of the inhibitory action of PG12 on the ADP platelet aggregatory influence persisted even after two hours following inactivation at 37 QC. Additional results obtained with isolated coronary arteries suggested the possible participation in the incubates, of a new compound with intereating biological properties. Indeed,the vascular relaxing effect of PGI2 (kept in either HRE, PRP or PPP) decreased progressively during the first half hour of incubation. This being transformed into s distinct enhancement of the coronary resting tone following the delivery of incubates of PGI2 obtained at the end of a longer period of permanence in human plasma but not in HRE. It is well known that, the b-keto-PGFq0( is the more common product of prostscyclin inactivation (5). Therefore, it was interesting to test the reaction to 6-keto-PGFqd following an incubation in human plasma. As previously reported no effect over coronary strips was observed during the first trisls (6). However after 60 minutes of incubation in plasma the compound produced a clear enhancement of the mechanical response of isolated bovine coronary preparations. It is plausible the existence in human plasma of enzymes able to transform PG12 or its hydrolytic product the 6-ketoPGFlh into another compound(s) having distinct and potent biological effects on coronary vessela and blood platelets. It has been recently reported that certain primary prostaglandins may be converted in some tissues into substances of different or even enhanced biological activity; rather then in non-active metsbolites. Examples of converting enzymes documented in several tissues include a prostsglsndin-E-9-ketoreductaae which transform prostaglandin E2 into PGF2& (7,8). Hoult and Moore reported a 9-hydroxy-dehydrogenase able to transform prostaglandin F24 into E2 in rat, rabbit and guinea pig kidneys (9). It has also been described in the rat kidney another 9-hydroxy-dehydrogenase that transform prostaglandin F2d metabolites into their corresponding E derivatives (IO). Furthermore, in recent reports it has been suggested that in liver the 6-keto-PGF,a is enzymatically converted into 6-ketoPGEl (11). The enzyme involved in this reaction could also be The 6-keto-PGE, has antiaggregathe 9-hydroxy-dehydrogenase. tory activity as well as PG12 (11,12,13~. On the other hand the 6-keto-PGEq contract isolated bovine curonary arteries in contrast to PG12 which is characterized by a relaxing influence (6, similar to that found in rabbit II>. We suggest that an enzyme, liver, may be present in human plasma and able to produce 6keto-PGEI. However it cannot be excluded the formation of another compound, such as PGE.1or 6-15-diketo-PGF,d , although this last one even able to contract bovine coronary arteries it lades the capacity to inhibit ADP induced plstelet aggregation (14).
914
JUNE
1980 VOL. 19 NO. 6
PROSTAGLANDINS
The recent study by Szczeklik et al. (151, infunding PC;12 to heal thy men, appears to be in keeping with our notion. Indeed, they have documented that only after one hour after the termination of prostacyclin infusion the protecting effect on ADP plaThis time is a too long period telet aggregation is finished. for the maintenance of PG12 activity. Indeed, Dusting et al.(fi) have reported that the half life of prostacyclin in extravasated dog blood is only of three minutes. Szcezeklik et al. (13) on the other hand, have attributed the prolonged action of PG12 on platelet aggregation to increased levels of cyclic AMP in platelet because after one hour the presence of proatacyclin in the aggregometer cuvette containing PAP is higly dubious. In view of our findings it is possible that even in PRP proatacyclin is no longer present, but it could exist a product of enzymatic transformation having bovine coronary contracting capacity as well as human blood platelet antiaggregatory activity.
REFERENCE-S I>
Sorda,E.S., M.A.Lazzari, M.F.Gimeno and A.L.Gimeno. Human Platelet Rich Plasma and Human Serum protects from inactivation the antiaggregatory capacity of proatacyclin like material (PGI2) produced by the rat stomach fundua. Proataglandina D.R.Morton, J.H.Kinner, R.R.Gorman, J.R.M.C. 2) Johnson,R.A., Guire, F.F.Sun,’ N.Whittaker, S.Bunting, J.A.Salmon, S.Moncada and J.R.Vane. The chemical characterization of proataglandin X (prostacyclin). Prostaglandins 12: 915, 1976. and M.A.Allen. Chemical stability 3) Cho,M.J. (PGI2) in aqueous solutions. Proataglandins
of prostacyclin 15: 943, 1978.
A.L.Gimeno and M.F.Gimeno. Frequency force 4) Sterin-Borda,L., relationship on isolated rat and guinea pig atria. Lffects of cholinergic and adrenergic receptor antagonists. Proc. Soc.Exp.Siol.Med. 145: 1151, 1974. S’.M:ncada and J.V.Vane. Arterial R.Gryglewaki, 5) Eunting,S., walls generate from prostaglandin endoperoxides a substance (proataglandin X) which relaxes strips of meaenteric and colliac arteries and inhibits platelet aggregation. Proataglandina 2: 897, 1976. 6) Dusting,G.J., proatacyclin
S.Mrrncada and J.R.Uane. Recirculation (PGI2) in the dog. fjr.J.Pharmac. 64:
Metabolism 7) Hamber,M. ancl U.Iaraelsson. in guinea-pig liver. I. Identification J.Giol.Chem. 245: 5107, 1970. 6)
of 315,
1970.
of proataglandin t2 of seven metabolites.
Lealie,C.A. and L.Levine. Evidence for the presence of a prostaglandin E2 9-ketoreductase in rat organs. Siochem. Siophys.Res.Commun. 2: 717, 1973.
JUNE 1980 VOL. 19 NO. 6
915
PROSTAGLANDINS
(9)
Hoult,J.R.S. and P.H.Moore. Enzyme in rabbit kidnet to prostaglandin verts prostaglandin F o( directly vitro. Br.J.Pharmac. _2_: 415 P, 1978. g.
cunE2 in
(IO)
Pace-Asciak,C. Prostaglandin ity in the adult rat kidney. way and some enzyme properties.
(II)
Wong,P.Y.H., K.lJ.Malik, F.F.Sun, W.H.Lee and J.C.McGiff. Hepatic metabolism of prostacyclin (PGI2) in the rabbit: formation of a potent inhibitor of platelet aggregation. Proc. 4th Int.Conference on Prostaglandins. Washington, p. 127, 1979.
(12)
9-hydroxydehydrogenase Identification, assay, J.biol.Chem.
activpath-
Lee.W.H., J.C.McGiff, R.W.Householder, F.F.Sun and P.Y.H. Wong. Inhibition of platelet aggregation by fi-keto-prostaa metabolite of 6-keto-prostaglandin glandin Eq (&HPGEl), Fq d (6-KPGF, d). Fed.Proc.Am.Soc.Exp.Biol. 38: 419, 1979.
(13)
Guilley,C.P., P.Y.H.Wong and J.C.McGiff. Hypotensive and renovascular actions of &keto-prostaglandin El a metabolite of prostacyclin. Eur.J.Pharmacol. 2: 273, 1979.
(14)
Gryglewaki,R.J. in “Advances in Endoperoxides,Prostacyclin and Tromboxane ResearchI’. 6th Course of the International School of Pharmacology. Erice, Italy, 2-3 September, 1979. In press.
(15)
Szczeklik,A., R.J.Gryglewski, R.Nizankowski, J.Musial, R. Pieton and J.Mruk. Circulatory and anti-platelet effects of intravenous prostacyclln in healthy men. Pharm.Res.Comm. -IO: 545, 1978.
916
JUNE 1980 VOL. 19 NO. 6
(PG12) INTO WHICH
CONTRACTS
+* *+ M.F.Glmeno+, L.Sterin-Borde , E.S.Borda+*, M.A.Lazzarl and A.L.Gimeno* Centro de Estudios Farmacolbglcos y de Principios Naturales (CEFAPRIN), CONICET, V. de Obligado 2490, 1428 Buenos Aires, ARGENTINA and Instituto de Investigaciones Hemato16gicas. Academia National de Mediclna. Pscheco de Melo 3081, 1425 Buenos Aires, ARGENTINA. ABSTRACT Prostacyclin (PG12) incubated In Human Platelet Rich Flasma (PAP); in Platelet Poor Plasma (PPP) or in Hrebs-RingerBicarbonate (HRB) during different periods of time on contractions of bovine coronary arteries and on the ADP platelet aggnzgative capacity of human PAP, were explored. It was documented that incubates In PRP or in PPP retain an antiaggregatory activity at higher levels and during a longer time than in HR6.h the other hand, PGI2 Incubates In KRB exhibited only a relaxing activity on Isolated bovine coronary arteries, whereas when incubated In PRP or in PPP presented a biphasic influence. 'The initial effects (evoked by incubates of 30 minutes) were distinctly relaxing but those obtained with later incubates (60150 minutes) stimulated clearly the resting basal tone of the arteries. The possibility that the human plasma might have an enzyme(s) able to transform prostacyclin into a more stable material with human antiaggregatory platelet function and bovine coronary Mailing
contracting Address:
capacity
is
A.L. GIMENCJ,M.D. V. de Gbligado 2490 1428 Buenos Aires, ARGENTINA
(1) This work has been supported National
discussed.
de Investigaclones
by Grant Clentiflcas
6630 from the Consejo y T&cnlcas de la
Repcbllca Argentina (CONICCT). * Senior Investigator, ** Junior
Investigator,
CONICET. CONICET.
INTRODUCTIUN Prostscyclin (FIG1 > is en unstable prostaglandin having a particularly short haff life. The menouremcnt of its rate of release from a tissue nnt only depends on the rate of production and diffusion out of the tissue but also on the ability
JUNE
1980 VOL. 19 NO. 6
907
PROSTAGLANDINS of the tissue and the incubation media to degrade it. We have recently shown that a PGI2-like material produced by the rat stomach fundus retains its antiaggregatory activity in human plasma or serum during a period of time significantly longer than in Hrebs solution (I). The fact that one of the biological activities of a PGI2-like substance persist in plasma or serum more than expected from stability studies of PGI2 in aqueous solutions (2,3) made us to suggest the possibility that the unstable prostaglandin material might be transformed into another compound also having antiaggregatory activity. The present experiments were designed in order to a8certain the validity of this hypothesis. Authentic PGI2 instead of PGI2-like material synthetized by rat stomach Pundus was used in order to avoid the possible role of other substances which might be present in the incubation medium. Prostacyclin was incubated in human platelet-rich plasma (PRP), human plateletpoor plasma (PPP) and Hrehs-Ringer-Bicarbonate (KRS) during different periods of time. The biological activity of the incubates were tested on contractions of isolated bovine coronary arteries and on the ADP induced platelet aggregation of human platelet-rich plasma. MATERIALAND METHODS (I)
The following materials were used: (a) Prostacyclin sodium salt (*Iwas dissolved in 0.05 M Tris buffer (pH 9) on the day of the experiment; 6-k&o-PGEq(*) was first dissolved in ethanol and the day to be used in aliquots of this solution were diluted with Hrebs solution and bketo-PGF&(*) was prepared directly in KREI. (b) PGI2 and &keto-PGFTC( were additioned to: KrebsRinger-Bicarbonate composed as reported elsewhere (4); to human platelet-rich plasma prepared by centrifuging the blood for 10 min at 200 g and to human platelet-poor plasma obtained by centrifuging the blood after the PRP had been removed at 15Ofl g for 30 min. The blood was obtained from donors which had not taken aspirin for at least 7 days and collected into tubes containing Na citrate 3.1% (9:l v.v.). PGI was also added to KRS containing human serum albumin (4 and 6%). The pH of HHH, PKP and PPP determined at room temperature was 0.0 * 0.1. The following test were performed: (a> Antiaggregatory sctivity. Prostacyclin in Tris buffer solution (16 ng in 16 ,ul) were added to 3 test tubes containing 400 ~1 of KHS, PRP and PPP respectively. Aliquots of 50 ul (with 2 ng of PGI2)from each different media were tested for antiaggregatory activity using 400 ul of PUP in the aggregometer cuvette. This dose of PGI2 added to the PRP inhibited ADP-induced platelet aggregatim by approximately 90%. The aggregation was triggered by ADP at 5 JJMfinal concentration. Control measurements were performed (II)
908
JUNE 1980 VOL. 19 NO. 6
PROSTAGLANDINS after adminietratinn of 50 ul of KRS, PAP or PPP without prostacyclin. Light transmission through the sample was recorded as a measurement of aggregation. The highest recorded value of light transmission following ADP addition was taken as the maximal ADP aggregation (100%) for that bath of PRP. Forthwith, the 3 media plus PGIz were incubated in a thermostatized bath at 37 oC and samples of 50 ul were taken at 30, 60, 90 and 120 min. The antiaggregatory activity of PG12 in the various media at different incubation times was expressed as per cent of maximal aggregation induced by ADP in control condition. b) Tonic changes of isolated bovine coronary arteries. Bovine hearts of freshly slaughtered animals were immersed in cold KRS and transported to the laboratory. The left descending anterior coronary artery and several of its primary branches were dissected out separated free of fat and cut hellically. Arterial strips were mounted in 6 different baths (numerated from 1 to 6) containing 20 ml of KRS mixed with 5% CO2 in oxygen at 37 QC and pH 7.4. The tissues were allowed to equilibrate for at least 90 min under a resting tension of 2 g. The mechanical changes of the preparation elicited by experimental additions were expressed as mg of tension above or under this reeting or basal tension (Cl tension in the figures). A force transducer (Statham Gold-Cell-U C 31 coupled to an ink writing oscilloqraph (San EI IS0 System) were used. At 0 time following equilibration 0.5 ug of PGI2 (in 0.1 ml of KRG, PRP or PPP) were additioned toleach tissue bath giving a final . After its effect was recorded the concentration of 25 ng.ml preparations were washed 3 times and allowed to recover their basal level at least for 15 minutes. PGIz incubated in KRB was inactivated at 37 W and aliquots of 0.1 ml were taken at 15, 30, GO, 90, 120 and 150 min. The samples corresponding to 15,6U and 120 min were tested on the coronary strip mounted in the bath number 1, whereas those of 30, 90 and 150 min in the strip mounted in the bath number 2. The same procedure was followed for PRP + PGI2 and PPP + PGIz using the arterial strips mounted in baths number 3, 4 and 5, 6 respectively. This procedure allowed enough time for the complete recovery of the basal tone after washings. Identical experiments were also performed employing G-keto-PGF# incubated during different periods in PPP. Furthermore in some preparations several concentrations of non-incubated 6keto-PGL 1 kjere tested. Experimental results were compared employing the Differences between means uere considered sigStudent t test. nificant-if
(*>
JUNE
p = 0.05
or
less.
Kindly provided by Dr. J.E.Pike; Kalamazon, Michigan, U.S.A.
1980 VOL. 19 NO. 6
The Upjnhn
Company,
909
PROSTAGLANDINS
RESULTS
results nf the inactivating PG12 at 37 QC in different media are summarized in figure 1. It can be seen that almost all the antiaggregatory activity of PGI2 disappeared after 60 min when diluted in KRB. On the other hand, more than 5fl%of this biological action was still present when PG12 was during the ssme period of time incubated in PRP or PPP. The most surprising finding was that after 120 min of incubation the antiaggregatory effect of PGI only diminished around 5U% both in PRP or PPP. Inasmuch as p1asma proteins could alter the chemical hydrolysis of PG12 experiments were done adding prostacyclin to KRB containing human serum albumin. It was noticed that albumin failed to influence the inactivation time of PG12. In order to search whether in the presence of plasma PGI2 was transformed into another material with biological activity on isnlated vessels, prostacyclin was incubated in different media (HUE, PAP or PPP) at 37 QC and then tested over isolated bovine coronary arteries (Figure 2, panel (a) show that in HHB the typical relaxing action of P&I2 decreased as the incubation time progresses disappearing at 60 min when the unstable prost+ glandin becames inactivated. Following this time the arterial strips did not respond with any mechanical change to the additions. It is interesting to point out that the same result was obtained measuring the antiaggregatory activity of PG12 incubated in KRD solution. On the other hand, when PG12 was kept in PRP or PPP the characteristic initial inhibitory action of PG12 was also seen, diminishing with time up to 30 min.However starting from the 60 min trial the tension of the bovine coronary artery commence to increase, reaching the maximal response with incubates of 150 min (figure 2 panels (b) and cc>. After this period of time the increment in tone induced by PGI2 incubated in PAP or PPP was sensible minor (results not shown in figures). Controls with human plasma without prostacyclin run at Cl time and after 120 min of incubation at 37 QC failed to induce mechanical responses. Figure 3 depicts tracings showing the changes in tension of coronary arterial strips recorded when PG12 or 6-keto-PGFld were incubated during different times and then delivered to the tissue bath. The arteries presented a lack of responsiveness to the addition of 6-keto-PGFlclin PPP up to 30 min, but after 120 min of incubation the tissue reacts with a clear increment of the basal tone (figure 3, middle tracing). The magnitud of contractions became similar to that evoked by PG12 incubated during equivalent periods in PPP or to that obtained with 15 to 25 ng of 6-ketn-PGE1 (figure 3, upper and lower tracings, respectively). The
DISCUSSION We have recently demonstrated that a PGI?-like material synthetized by the isolated rat stomach fundus conserved during a
910
JUNE 1980 VOL. 19 NO. 6
PROSTAGLANDINS
\ 9
30
Figure
1.
4
16)
60 TIME (min)
I
90
1
120
Inhibition of ADP platelet eggregation (meeaured a8 the lnverae of % light transmission) evoked by PGI2 (2 ng) Incubated during different perioda of time in Krebs-Ringer-Bicarbonate (KRB); Human Platelet-Rich Plaama@RP) or Human Platelet-Poor Pleama (PPP). Points and vertical bare = Means * SEM. Figure6 between parentheaea = number of experiments.
JUNE 1980 VOL. 19 NO. 6
911
PROSTAGLANDINS
+100 +
50
r
c
I I I
I I
PGI2+ KRB
(n-9)
- 100
G
l
100
+
50
E
PG12+ PRP (n=7)
0
E 3;
5
I-
-
50
t
-100 +lOO
+
50
PGI,+ PPP (n=8)
0
-
50
- 100
Figure
912
2.
0 15 30 60 90 120 150 INCUBATION TIME (min 1
Resting tone (in mg) of ieolated bovine coronary arteries a8 influenced by PGI (25 ng.ml’1) incubated during different perioda oi: time in HRB, PRP or PPP. Tension prior PG12 taken a8 reetlng basal 0 levele. Columns and vertical bare- Meana * SEM. For other detaila aee text and Fig. 1.
JUNE
1980 VOL. 19 NO. 6
PROSTAGLANDINS
JUNE 1980 VOL. 19 NO. 6
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longer time its platelet antiaggregatory capacity when incubated in human plasma or sera than when kept in HRE. In the present study, employing authentic prostacyclin, it was documented that no less than 50% of the inhibitory action of PG12 on the ADP platelet aggregatory influence persisted even after two hours following inactivation at 37 QC. Additional results obtained with isolated coronary arteries suggested the possible participation in the incubates, of a new compound with intereating biological properties. Indeed,the vascular relaxing effect of PGI2 (kept in either HRE, PRP or PPP) decreased progressively during the first half hour of incubation. This being transformed into s distinct enhancement of the coronary resting tone following the delivery of incubates of PGI2 obtained at the end of a longer period of permanence in human plasma but not in HRE. It is well known that, the b-keto-PGFq0( is the more common product of prostscyclin inactivation (5). Therefore, it was interesting to test the reaction to 6-keto-PGFqd following an incubation in human plasma. As previously reported no effect over coronary strips was observed during the first trisls (6). However after 60 minutes of incubation in plasma the compound produced a clear enhancement of the mechanical response of isolated bovine coronary preparations. It is plausible the existence in human plasma of enzymes able to transform PG12 or its hydrolytic product the 6-ketoPGFlh into another compound(s) having distinct and potent biological effects on coronary vessela and blood platelets. It has been recently reported that certain primary prostaglandins may be converted in some tissues into substances of different or even enhanced biological activity; rather then in non-active metsbolites. Examples of converting enzymes documented in several tissues include a prostsglsndin-E-9-ketoreductaae which transform prostaglandin E2 into PGF2& (7,8). Hoult and Moore reported a 9-hydroxy-dehydrogenase able to transform prostaglandin F24 into E2 in rat, rabbit and guinea pig kidneys (9). It has also been described in the rat kidney another 9-hydroxy-dehydrogenase that transform prostaglandin F2d metabolites into their corresponding E derivatives (IO). Furthermore, in recent reports it has been suggested that in liver the 6-keto-PGF,a is enzymatically converted into 6-ketoPGEl (11). The enzyme involved in this reaction could also be The 6-keto-PGE, has antiaggregathe 9-hydroxy-dehydrogenase. tory activity as well as PG12 (11,12,13~. On the other hand the 6-keto-PGEq contract isolated bovine curonary arteries in contrast to PG12 which is characterized by a relaxing influence (6, similar to that found in rabbit II>. We suggest that an enzyme, liver, may be present in human plasma and able to produce 6keto-PGEI. However it cannot be excluded the formation of another compound, such as PGE.1or 6-15-diketo-PGF,d , although this last one even able to contract bovine coronary arteries it lades the capacity to inhibit ADP induced plstelet aggregation (14).
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1980 VOL. 19 NO. 6
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The recent study by Szczeklik et al. (151, infunding PC;12 to heal thy men, appears to be in keeping with our notion. Indeed, they have documented that only after one hour after the termination of prostacyclin infusion the protecting effect on ADP plaThis time is a too long period telet aggregation is finished. for the maintenance of PG12 activity. Indeed, Dusting et al.(fi) have reported that the half life of prostacyclin in extravasated dog blood is only of three minutes. Szcezeklik et al. (13) on the other hand, have attributed the prolonged action of PG12 on platelet aggregation to increased levels of cyclic AMP in platelet because after one hour the presence of proatacyclin in the aggregometer cuvette containing PAP is higly dubious. In view of our findings it is possible that even in PRP proatacyclin is no longer present, but it could exist a product of enzymatic transformation having bovine coronary contracting capacity as well as human blood platelet antiaggregatory activity.
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Lealie,C.A. and L.Levine. Evidence for the presence of a prostaglandin E2 9-ketoreductase in rat organs. Siochem. Siophys.Res.Commun. 2: 717, 1973.
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Hoult,J.R.S. and P.H.Moore. Enzyme in rabbit kidnet to prostaglandin verts prostaglandin F o( directly vitro. Br.J.Pharmac. _2_: 415 P, 1978. g.
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Lee.W.H., J.C.McGiff, R.W.Householder, F.F.Sun and P.Y.H. Wong. Inhibition of platelet aggregation by fi-keto-prostaa metabolite of 6-keto-prostaglandin glandin Eq (&HPGEl), Fq d (6-KPGF, d). Fed.Proc.Am.Soc.Exp.Biol. 38: 419, 1979.
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Gryglewaki,R.J. in “Advances in Endoperoxides,Prostacyclin and Tromboxane ResearchI’. 6th Course of the International School of Pharmacology. Erice, Italy, 2-3 September, 1979. In press.
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Szczeklik,A., R.J.Gryglewski, R.Nizankowski, J.Musial, R. Pieton and J.Mruk. Circulatory and anti-platelet effects of intravenous prostacyclln in healthy men. Pharm.Res.Comm. -IO: 545, 1978.
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