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Contractile characteristics of the rat stomach strip and the rabbit aorta and mesenteric artery
Contractile Characteristics of the Rat Stomach Strip and the Rabbit Aorta and Mesenteric Artery
F. J. ZIJLSTRAAND J. E. VINCENT A comparison was made of the dose-response curves obtained in the measurement of the prostaglandin E- and thromboxane AZ-like activities using a cascade system consisting of a rabbit aorta strip and mesenteric artery and a rat fundus strip. The results indicate that the contractile properties of these tissues are different. In platelets during aggregation, the TxAJike activity was measured by bioassay; the amount of TxBz by a radioimmunological method. Key Words: bit mesenteric
Prostaglandin
E; Thromboxane
AZ; Rat stomach;
Rabbit aorta;
Rab-
artery
INTRODUCTION
In the determination of the biological activity of a number of substances, the effect on the contraction and relaxation of tissues is measured using the superfusion technique (Finkleman, 1930; Gaddum, 1953; Vane, 1964). Bioassays for both prostaglandins (PCs) and thromboxane A2 (TxAJ with the organ cascade have been described (Piper and Vane, 1969; Bunting et al., 1976). The PCE- and TxA,-like activities are determined by measuring the contractions of the rat stomach strip for the first and the rabbit aorta and mesenteric artery for the second effect. In our experiments, the PGE- and TxA,-like activities were determined in platelets after aggregation using a cascade system consisting of these three organs. It was found that the log-dose activity ratios for angiotensin II (A II), which is used as a reference substance in the determination of TxA,-like activity, did not form a straight line when plotted in the case of the rabbit aorta and mesenteric artery. For this reason, the contractile characteristics of the three organs were determined. In platelets, after aggregation, TxA2 is rapidly converted into the metabolite TxB,. A comparison was made between the magnitude of the TxA,-activity and the total amount of TxB, present as measured by a radioimmunological method. METHODS
AND
MATERIALS
Organ Cascade System In the bioassay, aortic strips and mesenteric arteries from male New Zealand albino rabbits and rat fundus strips were used. The contractions were measured From the Department of Pharmacology, Medical Faculty, Erasmus University Rotterdam, Rotterdam, The Netherlands. Address requests for reprints to Dr. J. E. Vincent, Department of Pharmacology, Medical Faculty, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. Received March 24, 1980; revised and accepted September 15, 1980.
1 Journal of Pharmacological 8 1981 Elsevier
North
Methods
Holland,
6, l-4
(1981)
Inc., 52 Vanderbilt
Avenue,
New York, NY 10017
Ol60-5402/81/0500001004$02,50
2
F. j. Zijlstra and J. E. Vincent
isotonically with Harvard heart/smooth muscle transducers and recorded. The strips were superfused at 2ml/min and 37°C with a gassed (95% 02, 5% COz) Krebs solution containing (mM): NaCl (118), KCI (4.7), CaCI, (2.5), MgS04 (1.18), KH,PO, (1.18), NaHC03 (25), and glucose (5.6). A mixture of the following antagonists was added to the solution (PM): atropine (O&I), phentolamine (0.08), sotalol (3.27), mepyramine (0.14), methysergide (O.lO), and indomethacin (2.80). The last named substance is used to inhibit the formation of PGs by the organs. In the measurement of the standard activities, cumulative dose-response curves were made. Platelet Aggregation Citrated blood was obtained from male volunteers who had not taken aspirin or other antiinflammatory drugs for at least a week. The blood was centrifuged for 5 min at 300 x g, and the supernatant platelet rich plasma (PRP) was collected. Saline was added to obtain a final concentration of 3 x IO8 platelets/ml. The platelet aggregation was determined as has been described before (Vincent et al, 1975). In the PRP, aggregation was induced by the addition of arachidonic acid to a final concentration of 1 mM. After stirring for 3 min, samples of 5, 10, 20, and 40 ~1 PRP were applied to the cascade system. Each sample was freshly prepared considering the short half-life of TxA2. TxB2 Determination The amount of TxB, formed in platelets during aggregation was measured using a protein-binding assay with a rabbit antithromboxane B2 serum. Aggregation was induced as described under platelet aggregation. After 3-min incubation, indo-
tion
mm contract
ion
J
7
mm contraction ._ _. ..
d \
,oo
2o01
100
log w ‘-2 RAT STOMACH
t-gAll RABBIT AORTA
200
I
P
2
8
20
log ng All MESENTERtC
ARTERY
FIGURE 1. Dose-effect ratios in the responses of the rat stomach strip to PCEp and of the rabbit aorta and mesenteric artery to angiotensin II. The substances were dissolved in Krebs buffer and applied to the top of the cascade.
Contractile
Characteristics of Isolated Organs
methacin (0.3 mM) was added to the sample. 100 ~1 was used in the test. After 20 hr incubation with the antiserum, the free antigen was adsorbed on charcoal and, after centrifugation, the supernatant was counted. The tritiated thromboxane B2 and the rabbit antithromboxane B2 serum were obtained from NEN. Materials
Prostaglandin E2 and arachidonic acid were obtained from Sigma, angiotensin from Ciba, and the antagonists from other commercial sources. RESULTS AND
II
DISCUSSION
The effects of PGE2 on the contractions of the rat stomach were determined. A linear relationship was found between the log-dose of PGE2 and the activity. The dose of A II was linearly related to the contractions of the rabbit aorta, whereas a log-dose log-contraction response is obtained in the case of the mesenteric artery (Figure 1). A comparison was made of the effects of A II and the TxA,-like activity of PRP on the rabbit aorta. The dose-response curves obtained have a different inclination. This indicates that in this system A II cannot be used as a reference substance for the measurement of TxA, activity (Figure 2). The dose-response curves are not significantly different in the case of the mesenteric artery. For this reason, the latter organ is better suited for the determination of the TxA,-like activity (Figure 3). An estimate of the amount of TxA, formed in the platelets is obtained by the determination of TxB, (Figures 2 and 3). These results indicate that the different contractile characteristics of the three tissues should be taken into account in the determination of the PGE and TxA,-like activities using this method.
25
0 0 t 0
10 I
1 100
20 I
30 I I 200
40 I
pl PRP I 300 ng TxB2
RABBIT AORTA
FIGURE 2. Dose-response curves of angiotensin II and PRP after aggregation on the rabbit aorta. C--O angiotensin II; u PRP. Values are expressed +SEM; n = 4.
3
4
F. J. Zijlstra and J. E. Vincent 0.5
*!
2 I
4 log 1
HIS
All -160 3 i --II 80 $ 8% 1” _f
- 20
- 10
5 1
10 I 50
1 100
20 I
I 200
MESEMERIC
FIGURE 3. Dose-response rabbit mesenteric artery. M n= 4.
40 IogpIPRP I log “9 1x82
ARTERY
curves of angiotensin angiotensin II; W
II and PRP after aggregation on the PRP. Values are expressed f SEM;
REFERENCES Bunting S, Moncada S, Vane JR (1976) The effects of prostaglandin endoperoxides and thromboxane A2 on strips of rabbit coeliac artery and certain other smooth muscle preparations. Br) Pbarmacol57:462-463P. Finkleman B (1930) On the nature of inhibition the intestine. / Physio/70:145-157.
in
Caddum JH (1953) The technique of superfusion. Br / Pharmacol Chemother 8:321-326. Piper PJ, Vane JR (1969) Releaseof additional factors
in anaphylaxis and its antagonism by anti-inflammatory drugs. Nature (Lond) 223:29-35. Vane JR (1964) The use of isolated organs for detecting active substances in the circulating blood. Br / Pharmacol Chemother 23:360-373. Vincent JE, Zijlstra FJ, Bonta IL (1975) The effect of non-steroid anti-inflammatory drugs, dibutyryl cyclic 3’5’-adenosine monophosphate and phosphodiesterase inhibitors on platelet aggregation and the platelet release reaction in normal and essential fatty acid deficient rats. Prostaglandins 10:899-911.
F. J. ZIJLSTRAAND J. E. VINCENT A comparison was made of the dose-response curves obtained in the measurement of the prostaglandin E- and thromboxane AZ-like activities using a cascade system consisting of a rabbit aorta strip and mesenteric artery and a rat fundus strip. The results indicate that the contractile properties of these tissues are different. In platelets during aggregation, the TxAJike activity was measured by bioassay; the amount of TxBz by a radioimmunological method. Key Words: bit mesenteric
Prostaglandin
E; Thromboxane
AZ; Rat stomach;
Rabbit aorta;
Rab-
artery
INTRODUCTION
In the determination of the biological activity of a number of substances, the effect on the contraction and relaxation of tissues is measured using the superfusion technique (Finkleman, 1930; Gaddum, 1953; Vane, 1964). Bioassays for both prostaglandins (PCs) and thromboxane A2 (TxAJ with the organ cascade have been described (Piper and Vane, 1969; Bunting et al., 1976). The PCE- and TxA,-like activities are determined by measuring the contractions of the rat stomach strip for the first and the rabbit aorta and mesenteric artery for the second effect. In our experiments, the PGE- and TxA,-like activities were determined in platelets after aggregation using a cascade system consisting of these three organs. It was found that the log-dose activity ratios for angiotensin II (A II), which is used as a reference substance in the determination of TxA,-like activity, did not form a straight line when plotted in the case of the rabbit aorta and mesenteric artery. For this reason, the contractile characteristics of the three organs were determined. In platelets, after aggregation, TxA2 is rapidly converted into the metabolite TxB,. A comparison was made between the magnitude of the TxA,-activity and the total amount of TxB, present as measured by a radioimmunological method. METHODS
AND
MATERIALS
Organ Cascade System In the bioassay, aortic strips and mesenteric arteries from male New Zealand albino rabbits and rat fundus strips were used. The contractions were measured From the Department of Pharmacology, Medical Faculty, Erasmus University Rotterdam, Rotterdam, The Netherlands. Address requests for reprints to Dr. J. E. Vincent, Department of Pharmacology, Medical Faculty, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. Received March 24, 1980; revised and accepted September 15, 1980.
1 Journal of Pharmacological 8 1981 Elsevier
North
Methods
Holland,
6, l-4
(1981)
Inc., 52 Vanderbilt
Avenue,
New York, NY 10017
Ol60-5402/81/0500001004$02,50
2
F. j. Zijlstra and J. E. Vincent
isotonically with Harvard heart/smooth muscle transducers and recorded. The strips were superfused at 2ml/min and 37°C with a gassed (95% 02, 5% COz) Krebs solution containing (mM): NaCl (118), KCI (4.7), CaCI, (2.5), MgS04 (1.18), KH,PO, (1.18), NaHC03 (25), and glucose (5.6). A mixture of the following antagonists was added to the solution (PM): atropine (O&I), phentolamine (0.08), sotalol (3.27), mepyramine (0.14), methysergide (O.lO), and indomethacin (2.80). The last named substance is used to inhibit the formation of PGs by the organs. In the measurement of the standard activities, cumulative dose-response curves were made. Platelet Aggregation Citrated blood was obtained from male volunteers who had not taken aspirin or other antiinflammatory drugs for at least a week. The blood was centrifuged for 5 min at 300 x g, and the supernatant platelet rich plasma (PRP) was collected. Saline was added to obtain a final concentration of 3 x IO8 platelets/ml. The platelet aggregation was determined as has been described before (Vincent et al, 1975). In the PRP, aggregation was induced by the addition of arachidonic acid to a final concentration of 1 mM. After stirring for 3 min, samples of 5, 10, 20, and 40 ~1 PRP were applied to the cascade system. Each sample was freshly prepared considering the short half-life of TxA2. TxB2 Determination The amount of TxB, formed in platelets during aggregation was measured using a protein-binding assay with a rabbit antithromboxane B2 serum. Aggregation was induced as described under platelet aggregation. After 3-min incubation, indo-
tion
mm contract
ion
J
7
mm contraction ._ _. ..
d \
,oo
2o01
100
log w ‘-2 RAT STOMACH
t-gAll RABBIT AORTA
200
I
P
2
8
20
log ng All MESENTERtC
ARTERY
FIGURE 1. Dose-effect ratios in the responses of the rat stomach strip to PCEp and of the rabbit aorta and mesenteric artery to angiotensin II. The substances were dissolved in Krebs buffer and applied to the top of the cascade.
Contractile
Characteristics of Isolated Organs
methacin (0.3 mM) was added to the sample. 100 ~1 was used in the test. After 20 hr incubation with the antiserum, the free antigen was adsorbed on charcoal and, after centrifugation, the supernatant was counted. The tritiated thromboxane B2 and the rabbit antithromboxane B2 serum were obtained from NEN. Materials
Prostaglandin E2 and arachidonic acid were obtained from Sigma, angiotensin from Ciba, and the antagonists from other commercial sources. RESULTS AND
II
DISCUSSION
The effects of PGE2 on the contractions of the rat stomach were determined. A linear relationship was found between the log-dose of PGE2 and the activity. The dose of A II was linearly related to the contractions of the rabbit aorta, whereas a log-dose log-contraction response is obtained in the case of the mesenteric artery (Figure 1). A comparison was made of the effects of A II and the TxA,-like activity of PRP on the rabbit aorta. The dose-response curves obtained have a different inclination. This indicates that in this system A II cannot be used as a reference substance for the measurement of TxA, activity (Figure 2). The dose-response curves are not significantly different in the case of the mesenteric artery. For this reason, the latter organ is better suited for the determination of the TxA,-like activity (Figure 3). An estimate of the amount of TxA, formed in the platelets is obtained by the determination of TxB, (Figures 2 and 3). These results indicate that the different contractile characteristics of the three tissues should be taken into account in the determination of the PGE and TxA,-like activities using this method.
25
0 0 t 0
10 I
1 100
20 I
30 I I 200
40 I
pl PRP I 300 ng TxB2
RABBIT AORTA
FIGURE 2. Dose-response curves of angiotensin II and PRP after aggregation on the rabbit aorta. C--O angiotensin II; u PRP. Values are expressed +SEM; n = 4.
3
4
F. J. Zijlstra and J. E. Vincent 0.5
*!
2 I
4 log 1
HIS
All -160 3 i --II 80 $ 8% 1” _f
- 20
- 10
5 1
10 I 50
1 100
20 I
I 200
MESEMERIC
FIGURE 3. Dose-response rabbit mesenteric artery. M n= 4.
40 IogpIPRP I log “9 1x82
ARTERY
curves of angiotensin angiotensin II; W
II and PRP after aggregation on the PRP. Values are expressed f SEM;
REFERENCES Bunting S, Moncada S, Vane JR (1976) The effects of prostaglandin endoperoxides and thromboxane A2 on strips of rabbit coeliac artery and certain other smooth muscle preparations. Br) Pbarmacol57:462-463P. Finkleman B (1930) On the nature of inhibition the intestine. / Physio/70:145-157.
in
Caddum JH (1953) The technique of superfusion. Br / Pharmacol Chemother 8:321-326. Piper PJ, Vane JR (1969) Releaseof additional factors
in anaphylaxis and its antagonism by anti-inflammatory drugs. Nature (Lond) 223:29-35. Vane JR (1964) The use of isolated organs for detecting active substances in the circulating blood. Br / Pharmacol Chemother 23:360-373. Vincent JE, Zijlstra FJ, Bonta IL (1975) The effect of non-steroid anti-inflammatory drugs, dibutyryl cyclic 3’5’-adenosine monophosphate and phosphodiesterase inhibitors on platelet aggregation and the platelet release reaction in normal and essential fatty acid deficient rats. Prostaglandins 10:899-911.