- Email: [email protected]
Quantitation by bioassay of mixtures of prostaglandins E2 and I2
Prostaglandins
and
Medicine
4: 171-176,
QUANTITATICN BYBIOASSAYOFMIXTURFS
1980
OF PROSTAGLAM)INS E2 AND I2
P. Lukacsko, A.M. Roberts, E.J. Messina, G. Raley. New York Medical College, Valhalla, N.Y. 10595
Department of Physiology
ABSTRACT
A bioassay method is described for the simultaneous identification and quantitation of small amounts of prostaglandins E2 and 12. Strips of rabbit coeliac and bovine coronary arteries were suspended within a camaon chamber surrounded by a heated water bath. The tissues were coupled individually to transducers to record their length. Using a superfusion flow rate of 2.0 ml/min the rabbit coeliac and bovine coronary arterial strips responded in a highly reproducible manner to threshold doses of 0.1 ng PGE2 and 1.0 ng PGI2, respectively. This techniqueismore sensitive than the conventional bioassay methods to quantify and differentiate between prostaglandins. INTRODUCTICN The cascade superfusion technique as originally described by Finkelman (1) and modified by others (2,3,4,5) for the detection of biologically active substances is currently in widespread use for the assay of prostaglandins (PGs). While an individual PG moiety can be assayed accurately by the use of several tissues the presence of mixtures of PGs complicates the identification and quantitation since bioassay tissues are selective rather than specific for these substances. In this communication we describe an improvement in the technique that provides a sensitive and stable system for the sirmltaneous assay of PGE2 and prostacyclin (PG12), two of the major biologically active metabolites of arachidonic acid (6,7). The rabbit coeliac and bovine coronary arterial strips are the most useful for this purpose because of their ability to discriminate between these two PGs. METHODS
Calf hearts obtained inmediately after slaughter were drained of blood and immersed in ice cold Rrebs bicarbonate solution (RBS). During transport to the laboratory, which took no longer than 1 hour, the solution containing the hearts was surrounded by ice. The distal portions of the anterior descending and circumflex coronary arteries were carefully dissected out and put into KBS kept on ice. Male New Zealand rabbits weighing approximately 2.5 kg were killed by a heavy blow to the head. The abdominal cavity was incised, 171
the coeliac artery was removed and placed into ice cold RBS. The vessels were subsequently cleaned of fat and adhering connective tissue. Cleaned coronary arteries were put over a Pasteur pipette whereas the coeliac artery was put over polyethylene tubing (PE 10) in order to cut the arteries at a close spiral with fine scissors. The coronary artery was cut into strips about 2-3 mm wide by 30 mm long and the coeliac artery into strips about l-l.5 mm wide by 20 nnn long. During these procedures care was taken to keep the tissues moist and to handle the vessels gently to avoid any unnecessary stretch. Coronary strips not used immediately were kept at 4°C in a closed container in EBS that had been gassed with 95% 02, 5% CO2. Each day the vessels were stored, fresh RBS gassed with 95% 02, 5% CO2 was added; in this way the coronary strips were found to display adequate responsiveness for up to three days. Rabbit coeliac arteries were obtained fresh daily. Ihe tissues were separately attached at one end to a common plexiglass stand, one above the other, and at the opposite end to individual transducers (Fig. 1).
Fig. 1. Schematic showing the position of the bioassay tissues within the enclosed chamber that is surrounded by a heated water bath. The tissues are . superfused at a constant rate with a gassed, pre-warmed Rrebs bicarbonate solution. All samples are added to the superfusion stream at a point just above the first tissue. The entire stand and tissues were contained within a chamber enclosed by a heated water bath. In this manner all the tissues were kept at a uniform temperature, even at low superfusion rates. The rabbit coeliac and bovine coronary artery strips were maintained at 1 and 2 grams tension, respectively, and length changes recorded by means of auxotonic transducers (liarvard386) coupled to a Beckman R611 Dynograph. The tissues were superfused at a rate of 2.0 ml/min with RBS at 37°C containing a mixture of inhibitors (8) and indomethacin (1 pg/ml)(9). The solution was continuously aerated with 95% 02 and 5% CO2 in order to maintain the pH at 7.4. After a four-hour equilibration period samples containing mixtures of PCE2 and PC12 were added to the superfusion stream just above the first tissue.
172
A stock solution of PGE2 was prepared by dissolving 1.0 mg in 0.1 ml 95% ethanol (v/v) and 0.9 ml of 2.0 mM Na2C03. Working solutions of PGE2 were prepared on the day of use by diluting aliquots of the stock solution with appropriate volumes of KRS. Prostacyclin was prepared fresh daily as a stock solution (1 mg/ml) by dissolving crystals of PGI2 in 1 M TRIS buffer (pH>9) and stored on ice. Subsequent dilutions were made with RBS and used iannediately. Neither the vehicles for these PGs nor 6-keto-PGFl (the stable metalluted with bolite of PGI2), in concentrations of up to 20 ng/ml when 8. appropriate aliquots of KEiS,affected the bioassay tissues. RESDLTS AND DISCUSSION Prostaglandin 12:caused relaxation of both rabbit coeliac and bovine coronary artery strips whereas PGE2 caused strips of rabbit coeliac artery to relax and those of bovine coronary artery to contract. In over 95% of the cases PGE2, in concentrations as low as 0.1 "g/ml, relaxed the rabbit coeliac artery in a highly reproducible and dose dependent fashion whereas the bovine coronary artery did not generally respond to PGE2 at concentrations less than 3 ng/ml (Fig. 2).
Rabbit Coeliac Artery
Bovine Coronary Artery
-
30 minutes
0.1
0.2
0.4
0.4
\
v
1.0
3.0
5.0
,
ng PGE 2/ml
Fig. 2. Dose dependent relaxation and contraction to PGE2 by rabbit coeliac and bovine coronary artery strips, respectively. Prostaglandins were injected into a superfusion stream of 2 ml/min at 37°C. Response of the coeliac strip to PGE2 is highly reproducible. The coeliac arterial strip is at least an order of magnitude more sensitive to PGE2 than the bovine coronary strip. In contrast, at least 95% of the bovine coronary strips relaxed in a reproducible and dose dependent manner to PG12 in concentrations as low as 1.0 ng/ml but 10 ng/ml or greater concentrations were generally required in order for the coeliac arterial strip to respond (Fig. 3). The rabbit coeliac artery strip is at least an order of magnitude more sensitive than the bovine coronary arterial strip to PGE2 whereas the coronary is about an order of magnitude more sensitive than the coeliac arterial strip to PGI This difference in sensitivity of the tissues allows for discrimination 2' between the two PGs.
The effects of PG12, when present in concentrations
173
Rabbit Coeliac Artery
--
Bovine Coronary Artery
lmin
30 minutes later
t
1.0
t 2.0
ng PGI2lml
Fig. 3. Rose dependent relaxation to ICI2 b y rabbit coeliac and bovine coronary artery strips. Response of the coronary strip to HZ12 is highly reproducible. The coronary arterial strip is at least an order of magnitude more sensitive to PGI2 than the coeliac arterial strip. that would affect the coeliac strip and therefore interfere with the accurate quantitation of PGE2, can be eliminated by acidification or more simply by placing the sample into boiling water for several minutes. This procedure will convert PG12 to the biologically less active 6-keto-FGFl, without affecting the more stable FGE2 moiety. The effect of ICE2 on the coronary strip opposes that of FGI2, however, the bovine coronary artery is more sensitive to the relaxing effects of FG12 than to the contractile effects of lJGE2and when both are present in equivalent concentrations the coronary strip responds preferentially to FGI2,- in this situation PGE2 increases the rate at which tension of the coronary strip returns to baseline but does not appreciably influence the absolute decrease in tension (Fig. 4).
Bovine Coronary Artery
ngPG/ml
LO E2 1.0 I2
5.0 E2
5.0 E2 +5.0 12
5.0 12
Fig. 4. Effects of PGE2, PG12 and a mixture of PGE2 and PGI2 on a bovine coronary artery strip superfused at 2.0 ml/min at 37°C. This tissue is more sensitive toPGI2 than to FGE2 and responds preferentially to PG12 when mixtures of these FGs are superfused. 174
The presence of PGF2o and PGD2 in the samples also does not interfere with-the aforementioned responses because the bioassay tissues are at least one order of magnitude less sensitive to these agents than to PGE2 and PGI2. Whereas threshold responses to PGs of individual strips of each of the two types of arteries are similar, they respond quite variably to increasing doses of PGs so that the response of each bioassay tissue to PG mixtures cannot be quantified by means of a dose response curve previously constructed from other coeliac or coronary arterial strips. Therefore the responses of the vascular strips to samples with unknown mixtures of PGs must be closely matched with responses elicited separately with known concentrations of PGE2 and PG12. In this way one can assay for PGs by bracketing the responses to the unknown sample between those of samples with known amounts of PGs. Since with this method no chemical manipulation is necessary it can be of advantage when correlating a physiological event with the production of the arachidonic acid metabolites, PGE2 and the highly unstable PG12. We therefore use this technique to assay PGE2 and PG12 in samples of tissue fluids, plasma or incubation mixtures and have found it to be more reliable and more sensitive than the conventional bioassay methods.
This work was supported by a grant from the American Heart Association. also thank the Upjohn Company for the supply of prostaglandins.
We
REFERENCES
1.
Finkelman B. On the nature of inhibition in the intestine. Journal of Physiology 70: 145, 1930.
2.
Gaddum JH. The technique of superfusion. British Journal of Pharmacology 8: 321, 1953.
3.
Vane JR. The use of isolated organs for detecting active substances in circulating blood. British Journal of Pharmacology 23: 360, 1964.
4.
Moncada S, Ferreira SH, Vane JR. Bioassay of prostaglandins and biologically active substances derived from arachidonic acid. In Advances in Prostaglandin and Thromboxane Research. Vol. 5. (JC Frb'liched) Raven Press, New York, 1978.
5.
Henman MC, Naylor IL, Leach GDH. A critical evaluation of the use of a cascade superfusion technique for the detection and estimation of biological activity. Journal of Pharmacological Methods 1: 13, 1978.
6.
Terragno AD, Crowshaw K, Terragno NA, McGiff JC. Prostaglandin synthesis by bovine mesenteric arteries and veins. Circulation Research 37: 176 1975.
7.
de Deckere E&4, Nugteren DH, Ten Hoor F. Prostacyclin is the major prostaa glandin released from the isolated perfused rabbit and rat heart. Nature 268: 160, 1977.
175
8.
Gilmore N, Vane JR, Wyllie JH. Nature 218: 1135, 1968.
Prostaglandins released by the spleen.
9.
Eckenfels A, Vane JR. Prostaglandins, oxygen tension and smooth muscle tone. British Journal of Pharmacology 45: 451, 1972.
176
and
Medicine
4: 171-176,
QUANTITATICN BYBIOASSAYOFMIXTURFS
1980
OF PROSTAGLAM)INS E2 AND I2
P. Lukacsko, A.M. Roberts, E.J. Messina, G. Raley. New York Medical College, Valhalla, N.Y. 10595
Department of Physiology
ABSTRACT
A bioassay method is described for the simultaneous identification and quantitation of small amounts of prostaglandins E2 and 12. Strips of rabbit coeliac and bovine coronary arteries were suspended within a camaon chamber surrounded by a heated water bath. The tissues were coupled individually to transducers to record their length. Using a superfusion flow rate of 2.0 ml/min the rabbit coeliac and bovine coronary arterial strips responded in a highly reproducible manner to threshold doses of 0.1 ng PGE2 and 1.0 ng PGI2, respectively. This techniqueismore sensitive than the conventional bioassay methods to quantify and differentiate between prostaglandins. INTRODUCTICN The cascade superfusion technique as originally described by Finkelman (1) and modified by others (2,3,4,5) for the detection of biologically active substances is currently in widespread use for the assay of prostaglandins (PGs). While an individual PG moiety can be assayed accurately by the use of several tissues the presence of mixtures of PGs complicates the identification and quantitation since bioassay tissues are selective rather than specific for these substances. In this communication we describe an improvement in the technique that provides a sensitive and stable system for the sirmltaneous assay of PGE2 and prostacyclin (PG12), two of the major biologically active metabolites of arachidonic acid (6,7). The rabbit coeliac and bovine coronary arterial strips are the most useful for this purpose because of their ability to discriminate between these two PGs. METHODS
Calf hearts obtained inmediately after slaughter were drained of blood and immersed in ice cold Rrebs bicarbonate solution (RBS). During transport to the laboratory, which took no longer than 1 hour, the solution containing the hearts was surrounded by ice. The distal portions of the anterior descending and circumflex coronary arteries were carefully dissected out and put into KBS kept on ice. Male New Zealand rabbits weighing approximately 2.5 kg were killed by a heavy blow to the head. The abdominal cavity was incised, 171
the coeliac artery was removed and placed into ice cold RBS. The vessels were subsequently cleaned of fat and adhering connective tissue. Cleaned coronary arteries were put over a Pasteur pipette whereas the coeliac artery was put over polyethylene tubing (PE 10) in order to cut the arteries at a close spiral with fine scissors. The coronary artery was cut into strips about 2-3 mm wide by 30 mm long and the coeliac artery into strips about l-l.5 mm wide by 20 nnn long. During these procedures care was taken to keep the tissues moist and to handle the vessels gently to avoid any unnecessary stretch. Coronary strips not used immediately were kept at 4°C in a closed container in EBS that had been gassed with 95% 02, 5% CO2. Each day the vessels were stored, fresh RBS gassed with 95% 02, 5% CO2 was added; in this way the coronary strips were found to display adequate responsiveness for up to three days. Rabbit coeliac arteries were obtained fresh daily. Ihe tissues were separately attached at one end to a common plexiglass stand, one above the other, and at the opposite end to individual transducers (Fig. 1).
Fig. 1. Schematic showing the position of the bioassay tissues within the enclosed chamber that is surrounded by a heated water bath. The tissues are . superfused at a constant rate with a gassed, pre-warmed Rrebs bicarbonate solution. All samples are added to the superfusion stream at a point just above the first tissue. The entire stand and tissues were contained within a chamber enclosed by a heated water bath. In this manner all the tissues were kept at a uniform temperature, even at low superfusion rates. The rabbit coeliac and bovine coronary artery strips were maintained at 1 and 2 grams tension, respectively, and length changes recorded by means of auxotonic transducers (liarvard386) coupled to a Beckman R611 Dynograph. The tissues were superfused at a rate of 2.0 ml/min with RBS at 37°C containing a mixture of inhibitors (8) and indomethacin (1 pg/ml)(9). The solution was continuously aerated with 95% 02 and 5% CO2 in order to maintain the pH at 7.4. After a four-hour equilibration period samples containing mixtures of PCE2 and PC12 were added to the superfusion stream just above the first tissue.
172
A stock solution of PGE2 was prepared by dissolving 1.0 mg in 0.1 ml 95% ethanol (v/v) and 0.9 ml of 2.0 mM Na2C03. Working solutions of PGE2 were prepared on the day of use by diluting aliquots of the stock solution with appropriate volumes of KRS. Prostacyclin was prepared fresh daily as a stock solution (1 mg/ml) by dissolving crystals of PGI2 in 1 M TRIS buffer (pH>9) and stored on ice. Subsequent dilutions were made with RBS and used iannediately. Neither the vehicles for these PGs nor 6-keto-PGFl (the stable metalluted with bolite of PGI2), in concentrations of up to 20 ng/ml when 8. appropriate aliquots of KEiS,affected the bioassay tissues. RESDLTS AND DISCUSSION Prostaglandin 12:caused relaxation of both rabbit coeliac and bovine coronary artery strips whereas PGE2 caused strips of rabbit coeliac artery to relax and those of bovine coronary artery to contract. In over 95% of the cases PGE2, in concentrations as low as 0.1 "g/ml, relaxed the rabbit coeliac artery in a highly reproducible and dose dependent fashion whereas the bovine coronary artery did not generally respond to PGE2 at concentrations less than 3 ng/ml (Fig. 2).
Rabbit Coeliac Artery
Bovine Coronary Artery
-
30 minutes
0.1
0.2
0.4
0.4
\
v
1.0
3.0
5.0
,
ng PGE 2/ml
Fig. 2. Dose dependent relaxation and contraction to PGE2 by rabbit coeliac and bovine coronary artery strips, respectively. Prostaglandins were injected into a superfusion stream of 2 ml/min at 37°C. Response of the coeliac strip to PGE2 is highly reproducible. The coeliac arterial strip is at least an order of magnitude more sensitive to PGE2 than the bovine coronary strip. In contrast, at least 95% of the bovine coronary strips relaxed in a reproducible and dose dependent manner to PG12 in concentrations as low as 1.0 ng/ml but 10 ng/ml or greater concentrations were generally required in order for the coeliac arterial strip to respond (Fig. 3). The rabbit coeliac artery strip is at least an order of magnitude more sensitive than the bovine coronary arterial strip to PGE2 whereas the coronary is about an order of magnitude more sensitive than the coeliac arterial strip to PGI This difference in sensitivity of the tissues allows for discrimination 2' between the two PGs.
The effects of PG12, when present in concentrations
173
Rabbit Coeliac Artery
--
Bovine Coronary Artery
lmin
30 minutes later
t
1.0
t 2.0
ng PGI2lml
Fig. 3. Rose dependent relaxation to ICI2 b y rabbit coeliac and bovine coronary artery strips. Response of the coronary strip to HZ12 is highly reproducible. The coronary arterial strip is at least an order of magnitude more sensitive to PGI2 than the coeliac arterial strip. that would affect the coeliac strip and therefore interfere with the accurate quantitation of PGE2, can be eliminated by acidification or more simply by placing the sample into boiling water for several minutes. This procedure will convert PG12 to the biologically less active 6-keto-FGFl, without affecting the more stable FGE2 moiety. The effect of ICE2 on the coronary strip opposes that of FGI2, however, the bovine coronary artery is more sensitive to the relaxing effects of FG12 than to the contractile effects of lJGE2and when both are present in equivalent concentrations the coronary strip responds preferentially to FGI2,- in this situation PGE2 increases the rate at which tension of the coronary strip returns to baseline but does not appreciably influence the absolute decrease in tension (Fig. 4).
Bovine Coronary Artery
ngPG/ml
LO E2 1.0 I2
5.0 E2
5.0 E2 +5.0 12
5.0 12
Fig. 4. Effects of PGE2, PG12 and a mixture of PGE2 and PGI2 on a bovine coronary artery strip superfused at 2.0 ml/min at 37°C. This tissue is more sensitive toPGI2 than to FGE2 and responds preferentially to PG12 when mixtures of these FGs are superfused. 174
The presence of PGF2o and PGD2 in the samples also does not interfere with-the aforementioned responses because the bioassay tissues are at least one order of magnitude less sensitive to these agents than to PGE2 and PGI2. Whereas threshold responses to PGs of individual strips of each of the two types of arteries are similar, they respond quite variably to increasing doses of PGs so that the response of each bioassay tissue to PG mixtures cannot be quantified by means of a dose response curve previously constructed from other coeliac or coronary arterial strips. Therefore the responses of the vascular strips to samples with unknown mixtures of PGs must be closely matched with responses elicited separately with known concentrations of PGE2 and PG12. In this way one can assay for PGs by bracketing the responses to the unknown sample between those of samples with known amounts of PGs. Since with this method no chemical manipulation is necessary it can be of advantage when correlating a physiological event with the production of the arachidonic acid metabolites, PGE2 and the highly unstable PG12. We therefore use this technique to assay PGE2 and PG12 in samples of tissue fluids, plasma or incubation mixtures and have found it to be more reliable and more sensitive than the conventional bioassay methods.
This work was supported by a grant from the American Heart Association. also thank the Upjohn Company for the supply of prostaglandins.
We
REFERENCES
1.
Finkelman B. On the nature of inhibition in the intestine. Journal of Physiology 70: 145, 1930.
2.
Gaddum JH. The technique of superfusion. British Journal of Pharmacology 8: 321, 1953.
3.
Vane JR. The use of isolated organs for detecting active substances in circulating blood. British Journal of Pharmacology 23: 360, 1964.
4.
Moncada S, Ferreira SH, Vane JR. Bioassay of prostaglandins and biologically active substances derived from arachidonic acid. In Advances in Prostaglandin and Thromboxane Research. Vol. 5. (JC Frb'liched) Raven Press, New York, 1978.
5.
Henman MC, Naylor IL, Leach GDH. A critical evaluation of the use of a cascade superfusion technique for the detection and estimation of biological activity. Journal of Pharmacological Methods 1: 13, 1978.
6.
Terragno AD, Crowshaw K, Terragno NA, McGiff JC. Prostaglandin synthesis by bovine mesenteric arteries and veins. Circulation Research 37: 176 1975.
7.
de Deckere E&4, Nugteren DH, Ten Hoor F. Prostacyclin is the major prostaa glandin released from the isolated perfused rabbit and rat heart. Nature 268: 160, 1977.
175
8.
Gilmore N, Vane JR, Wyllie JH. Nature 218: 1135, 1968.
Prostaglandins released by the spleen.
9.
Eckenfels A, Vane JR. Prostaglandins, oxygen tension and smooth muscle tone. British Journal of Pharmacology 45: 451, 1972.
176