thromboxane B2 ratio in vascular and lung tissue

THROMBOSTS RESEARCH 30; 643-650, 1983 0049-3848183 $3.00 + .OO Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.

6-KETO-PROSTAGLANDIN F,,/THROMBOXANE B2 RATIO IN VASCULAR AND LUNG TISSUE Pia Saldeen and Tom Saldeen Department of Experimental Research, MalmijGeneral Hospital, University of Lund, Sweden and Institute of Forensic Medicine, University of Uppsala, Sweden.

(Received 27.12.1982; Accepted in revised form 22.3.1983 by Editor G. Myllyll)

.ABSTRACT In vitro production of 6-keto-PGFlcland thromboxane B2 (TxB2) was determined by radioimmunoassay. The 6-keto-PGF,o/TxB2 ratio varied between 5 and 41 in vascular tissue and between-O.5 and 6 in lung tissue from various species. There was a correlation between the production of 6-keto-PGFlo and TxB2; the correlation coefficient being about 0.6. The production of 6-keto-PGF and TxB2 was stimulated by arachidonic acid and inhibited by indomet '4acin. The 6-keto-PGF /TxB ratio was increased by a thromboxane synthetase inhibitor and'vari&s peptides, e.g. DDAVP and bradykinin, and was decreased by arachidonic acid. It is suggested that the balance between 6-keto-PGFla and TxB production in the vessel wall could be of importance in certain pathozogical conditions, e.g. thrombus formation.

INTRODUCTION

Thromboxane A2 (TxA2) is a vasoconstrictor and an inducer of platelet aggregation, and prostacyclin (PG12) is a vasodilator and an inhibitor of platelet aggregation. It has been postulated that the balance between the amount of TxA2 formed by platelets and the amount of PG12 formed by blood vessels is critical for thrombus formation (3), on the basis that generation of PG12 may underly the ability of normal vessels to resist platelet adhesion. In a few investigations production of TxA2 by blood vessels (4-8) and lung tissue (1,8-10) has been reported. Production of PG12 and TxA2 can be determined by radioimmunoassay of their stable hydrolysis products 6-keto-PGFla and thromboxane B2 (TxB2). The present study concerns the 6-keto-PGF /ti,B2ratio in vascular and lung tissue from uence of various substances on this ratio. different species and the infia Key words: Prostacyclin, thromboxane, vascular tissue, lung tissue. 643

644

6-KETO-PgF1

/TxB2 RATIO

vo1.30, No.6

MATERIAL AND METHODS Vascular tissue. As a rule 0.5-l cm long vascular segments were used. A superficial vein segment was obtained from the back of the hand of patients. These specimens were stored at -7O'C for l-2 months until examined. No other specimens were frozen before examination. Macroscopically normal coronary arteries were obtained from autopsy cases within 8 hours after death. Bovine mesenteric arteries were provided by a local abattoir within 30 minutes of slaughtering. Carotid and femoral arteries and jugular veins were obtained from sheep within 30 minutes after death. Aortae and inferior vena cavae were obtained from rabbits and aortae from rats within 30 minutes after death. The vessels were carefully cleaned from blood by superfusion with saline. As a rule 100 mg tissue was used. The average weight of the hand vein specimens from patients was about 10 mg (range 5-15 mg). Lung tissue was taken from rats, rabbits and guinea pigs. The pulmonary vasculature was cleaned from blood by infusion of saline into the pulmonary artery. About 100 mg of lung tissue was used. Each lung was cut into 15 pieces. Incubation of the tissues. The tissues were incubated in 1 ml (phosphate buffered saline) pH 7.4, in polypropylene test tubes at rule for 10 or 30 minutes. The samples were gently agitated during in a shaking water bath. The tissue was removed and the buffer was stored at -7OOC until examined.

of PBS 37'C, as a incubation immediately

LIeterminationof PGI:,and TxA,. The stable hydrolysis products of PG12 and TxA2, 6-keto-PGF1, and TxB2, respectively, were determined by radioimmunoassay, using tritiated antigen and antirabbit antiserum as antibody (RIA Kits, New England Nuclear). The TxB2 antibody cross-reacts about 10 X with PGD , .0.2 % with PGE2 and less than 0.2 X with PGA2, PGFSa%annti-klc;~-PGFla.Tie 6keto-PGFla antibody cross-reacts 7.8 % with PGFla, 1, 2.7 % with PGF2,, 2 W with PGE2, less than 0.3 % with PGA1 and less than 0.1 X with PGA2 and TxB2. Each sample was assayed in duplicate. Of the substances used in the present investigation, indomethacin, UK 37, 248,DDAVP, dipyridamole, peptide 6A and bradykinin at the concentrations used did not disturb the RIAs. It has earlier been shown that a high concentration of arachidonic acid (6 x 10q5 M) to a certain extent disturbs the TxB2 RIA (11). When low concentrations of TxB2 were measured false high values were obtained in the presence of arachidonic acid, and when high concentrations of TxB2 (>50 pg/ml) were measured false low values were obtained in the presence of arachidonic acid (11). These findings were confirmed in the present investigation and high concentrations of arachidonic acid also disturbed the 6-keto-PGFlc(RIA. If high concentrations of arachidonic acid still were present during the measurement of the samples the values presented may thus be falsely low. Materials. Des-amino-D-arginine vasopressin (DDAVP) was a gift from Ferring AB, MalmS. Dipyridamole was a gift from Boehringer Ingelheim, BRD. The vasoactive peptide 6A (Ala-Arg-Pro-Ala-Lys) (12) was kindly synthesized by Ferring AB. Bradykinin was bought from Sigma, St. Louis, USA. Indomethacin (Confortid @) was a gift from Dumex, Sweden. UK 37, 248 (4-[2-(lll-imidazol-lyl) ethoxy] benzoic acid hydrochloride), a thromboxane synthetase inhibitor, was kindly supplied by Dr H.M. Tyler, Pfizer, England. Arachidonic acid was dissolved in methanol and stored in the dark as a 10 mg/ml solution. All other substances were dissolved in saline. RESULTS The production of 6-keto-PGFlcland TxB2 in vascular and lung tissue from various species is presented in Table I. Thromboxane production was found to

6-KETO-PgF1 /TxB2 RATIO

vo1.30, No.6

645

TABLE I Production of 6-keto-PGF1 and TxB2 in vascular and lung tissue from various species. R = 6-ke?!o-PGFI/TxB2 ratio.n = number of specimens. Incubation time 10 minutes. Meana? S.D. n

Human coronary astery

4

6-keto-PGFla

TxFi2

(pg/mg/min)

(pg/mg/min)

4.7 +

2.6

0.8 2 0.1

R

5.9

20

62

2 36

2.0 +_0.6

41

Bovine mesenteric artery

4

27

2 10

1.0 2 0.2

27

Sheep carotid artery

Human hand vein

5

17

t 11

1.8 $ 0.7

Sheep jugular vein

5

14

+

1.3 2 0.4

11

Rabbit aorta

3

29

2 11

0.8 f 0.5

36

Rabbit vena cava

3

11

2

2.0 2 0.6

Rat aorta

8

104

Guinea pig lung

6

10

2

Rabbit lung

5

30

+ 20

24

24

+

Rat lung

6.0

5.1

!:68 2.5

6.0

4.3 + 2.0

9.4

5.5 24

19

+ 7.4

0.5

12

2 4.5

2.5

4.0 2 1.2

5.9

loot3-t3- keto-P(SF1, I&mg

Fig 1 Correlation between production of 6-keto-PGFI and of TxB2 in sheep ca?otid arteries. The vessels were incubated for different periods (lo-30 min).

50(3-

0

20

40

60

80 Tx62 vglmg

646

6-KETO-PgF1

/TxB2 RATIO

vo1.30, No.6

occur in all studied tissues. The ratio of 6-keto-PGF,o to TxB2 varied between 5 and 41 in vascular tissue and between 0.5 and 6 in lung tissue from various species. The correlation coefficient between the production of the two substances was 0.64 for sheep carotid artery (Fig 1) and 0.63 for rat lung tissue. The only organ which produced more TxB2 than 6-keto-PGF,o was guinea pig lung (Table I). The time-dependent formation of the two substances and the inhibition of this formation by indomethacin, added in vitro, in rat lung are shownin Fig 2. Values in indomethacin-treated specimens are basal levels, whereas the difference between untreated and indometahcin-treated values represents de novo synthesis. A thromboxane synthetase inhibitor, UK 37, 248, both when infused intravenously and when added in vitro, reduced the production of TxB2 and greatly increased the 6-keto-PGFl,/TxB2 ratio (Tables II and III). Arachidonic acid increased the production of both substances and greatly decreased the 6keto-PGF,o/TxB2 ratio (Table III). DDAVP, dipyramidole, peptide 6A and bradykinin, added in vitro to lung tissue, all increased the 6-keto-PGFlo/TxB2 ratio (Fig 3), as a result of increased production of 6-keto-PGFla and unchanged synthesis of TxB2. The concentrations presented are the lowest concentrations for each substance giving significant increase of 6-keto-PGFlo!compared to controls. The values for 6keto-PGFla were 271277 pgfmg in the control group; 390287 with DDAVP, 5532 120 with dipyridamole, 477?141 with peptide 6A and 6952166 with bradykinin. DISCUSSION The present evidence that TxA2 is produced by blood vessels is based on the specificity of the radioimmunoassay for TxB and the inhibition caused by the specific thromboxane synthetase inhibitor. 1t cannot be ruled out that some of the TxA2 production in this study occurred in platelets trapped on the vessel wall or in the lungs. However, since we cleaned the specimens carefully from blood and since it has been shown recently not only by radioimmunoassay but also by two-dimensional thin layer chromatography that bovine aortic endothelial cells (13,14) produce TxA2 we believe the vessel wall to be the major producer of TxA2. The PG12/TxA2 ratio reported for endothelial cells, namely 5:l to 1O:l is lower than the ratio for bovine mesenteric artery in our investigation. One explanation for this difference may be the fact that smooth muscle cells seem to produce PGI2 but not TxA2 (14). (141,

In our lung specimens the vessels might have been the main source of PG12 and TxA2, since rabbit lung parenchyma has been reported not to produce these substances (6). Intrapulmonary arteries produce more TxA2 than extrapulmonary arteries (6), which may be one explanation for the lower PG12/TxA2 ratio in pulmonary than in vascular tissue observed in the present investigation. Our observations that guinea pig lung produces larger amounts of TxA2 than rat lung and that DDAVP, dipyridamole, bradykinin and peptide 6A stimulate PG12 production confirm earlier findings (10, 15-18). The values for the superficial veins from the hands of patients might not be directly comparable to those from the other tissues studied, since these vessels were frozen before examination. Maurer et al. (5) found a slight increase in 6-keto-PGFl b cerebral microvessels but no change in TxB2 due to freezing. The higher Feve1 of 6-keto-PGF,o was found to be due to higher basal whereas the amount synthesized during the experiment ~~~e~S,i~~t~,k',:~U%~~l~~). However, it has been reported by others that freezing and thawing do not interfere with the 6-keto-PGF,o and TxB2 determina-

vo1.30, No.6

6-KETO-PgFl

/TxB2 RATIO

647

TABLE II Effect of intravenously infused indomethacin, 4 mg/kg b.w., or a -thromboxane synthetase inhibitor, UK 37, 248, 4 mg/kg b.w., on production of 6-keto-PGFla and TxB2 in rat lung and rat aorta. The substances were infused 30 minutes before killing the animals. R = 6keto-PGFr,/TxBn ratio. n = number of specimens. Incubation time 10 minutes, Mean f S.D. *Significantly different from controls. n

6-keto-PGFlo

R

T32

(pg/mg/min)

(pg/mg/min)

32.2 f 3.6

5.1 + 0.9

6.3

Lung

Controls

11

Indomethacin

9

1.4 + 1.1*

1.4 + 0.8*

1

UK 37, 248

9

20.4 + 5.0"

1.2 ?;0.5*

17

4.3 + 2.0

24

Aorta Controls

8

Indomethacin

6

28.5 + 18.6X

1.5 ?r 1.0

19

UK 37, 248

6

65.7 f 34.1

1.4 2 0.7*

47

104

+ 68

TABLE III Effect of in vitro added indomethacin (200 pM), UK 37, 248 (200 PM) and arachidonic acid (200 pM)(finalconcentrations)on production of 6-keto-PGFlo and TxB2 in human coronary artery or rat lung, R = 6-keto-PGF&TxBz ratio. n = number of specimens. Incubation time 10 minutes: Mean + S.D. *Significantly different from controls. n

6-keto-PGFra

TxB2

(pg/mg/min)

Ypg/mg/min)

R

Human coronary artery Controls

4

4.7 + 0.6

0.8 * 0.1

6

Indomethacin

4

2.2 + 0.5*

0.3 i:0.1*

7

Arachidonic

4

15

+9

7.7 f 1.9*

1.9

13

16

f 9.0

2.7 f.2.0

6.0

1.2 t 0.5"

5.2

Rat lung Controls Indomethacin

9

UK 37, 248

4

40

f 6.2"

Arachidonic acid

7

51

+ lo*

6.2 f 4.3*

1.3 f 0.8 29

+ 11*

31 1.8

6-KETO-PgFl

648

vo1.30, No.6

/TxB2 EAT10

pg/mg 300.

200-

0 6-Keto-PGF1

a , controls

0 6-Keto-PGF1

a , indomethacin

0 TxB2, ??TxB2

controls , indomethacin

loo-

I 2

I 20

10

minutes

Fig 2 Time-dependent production of 6-keto-PGF,, and of TxB2 in rat lung tissue and effect of in vitro added indomethacin (200 pM,final concentration).

tions in human umbilical vessels (7).

We are currently studying the 6-keto-PGF,o/TxB2 ratio in veins from patients with recurrent venous thrombosis, in coronary arteries from patients with ischemic heart disease and in the pseudointima of vascular grafts. Preliminary results have shown a decreased ratio in these conditions, which may be due to inhibition of endothelial PG12 synthetase by, for example, lipid peroxides, and secondary stimulation of the thromboxane synthetase pathway, and could be a contributory cause of thrombus formation. In addition, a disturbance of the normal PGI,/TxA2 ratio in the pulmonary vascular bed might be of importance in pulmonary thromboembolism and in respiratory insufficiency after trauma and sepsis - the microembolism syndrome.

vo1.30,

No.6

6-KETO-PgFl

Cl

Controls

!a

DDAVP

am

Dipyridamole

? ?Peptide

1 O-gM 2x1 0w5M

6A 2x 10m4M

Bradykinin ??

649

/TxB2 RATTO

Ratio 1 10

2x 1 O-6M

Fig 3 Effect of DDAVP, dipyridamole, peptide 6A and bradykinin, added in vitro, on the 6-keto-PGF /TxB ratio in lung tissue. Incubation time 10 minutes. The c&ent?ations of the substances are final concentrations.

REFERENCES 1.

Hamberg, M., Svensson, J. and Samuelsson, B.: Thromboxanes: A new group of biologically active compounds derived from prostaglandin endoperoxi1975. des. Proc. Natl. Acad. Sci. 72, 2994-2998,

2.

Moncada, S., Gryglewski, R., Bunting, S. and Vane, J.R.: An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature (Lend.) 263,663-665, 1976.

3.

Vane, J.R., Bunting, S. and Moncada, S.: Prostacyclin in physiology and pathophysiology. Internat. Rev. Exp. Pathol. 23, 161-207, 1982.

4.

Tuvemo, T., Strandberg, K., Hamberg, M. and Samuelsson, B.: Formation and action of prostaglandin endoperoxides in the isolated human umbilical artery. Acta physiol. stand. 96, 145-149, 1976.

6-KETO-PgFl

650

vo1.30, No.6

/TxB2 RATIO

5.

Maurer, P., Moskowits, M-A., Levine, L. and Meland, E.: The synthesis of prostaglandins by bovine cerebral microvessels. BostagZandins and Medicine 4, 153-161, 1980.

6.

Salzman, P.M., Salmon, J.A. and Moncada, S.: Prostacyclin and thromboxane A synthesis by rabbit pulmonary artery. J. Pharmacol. Ep. Ther. 215, 220-247, 1980.

7.

MikilP, U.M., Wahlberg, L., Viinikka, L. and Ylikorkala, 0.: Regulation of prostacyclin and thromboxane production by human umbilical vessels: The effect of estradiol and progesterone in a superfusion model. Prosta-

glandins,Leukotrienesand Medicine, 8. 9.

10.

8,

115-124,

1982.

Saldeen, P., Carlin, G. and Saldeen, T.: In vitro synthesis of thromboxane A2 and prostacyclin. Haemostasis 12, 56, 1982. Dawson, W., Booth, J.R., Cockerill, A.F., Mallen, D.N.B. and Osborne, D.J.: Release of novel prostaglandins and thromboxanes after immunologi1976. cal challenge of guinea pig lung. Nature (London) 262, 697-702, Boyd, J.A. and Eling, T.E.: Prostaglandin release and the interaction of platelets with the pulmonary vasculature of rat and guinea pig. Thrombosis

Research

19, 239-248,

1980.

11. Andrassy, K., Koderisch, J., Kern, B and Ritz, E.: Does arachidonic acid interfere with measurements of thromboxane (TxB2)? Thromb. Haemostas.

(Stuttgart)

47, 185,

1982.

12. Belew, M., Gerdin, B., Lindeberg, G., Porath, J., Saldeen, T. and Wallin, R .: Structure-activity relationships of vasoactive peptides derived from fibrin or fibrinogen degraded by plasmin. Biochim. Biophys. Acta. 621,

169-178,

1980.

13. Levine, L. and Alam, I.: Arachidonic acid metabolism by cells in culture: Analyses of culture fluids for cyclooxygenase products by radioimmunoassay before and after separation by high pressure liquid chromatography.

Prostaglandins

and Medicine

3, 295-304,

1979.

14. Ingerman- Wojenski, C., Silver, M.J., Smith, J.B. and Macarack, E.: Bovine endothelial cells -inculture produce thromboxane as well as prostacyclin.

J. CZin. Invest.

67, 1292-1296,1981.

15. Belch, J.J.F., Small, M., McKenzie, F., Hill, P.A., Lowe, G.D.O., McIntyre, D.F., Forbes, C.D. and Prentice, C.R.M.: DDAVP stimulates prostacyclin production. Thromb. Haemostas. (Stuttgart) 47, 122-123, 1982. 16. Jackson, C.A., Greaves, M. and Preston, F.E.: A study of stimulation of human venous prostacyclin synthesis of dipyridamole. Thrombosis Research,

27, 563-573,

1982.

17. Mullane, K.M., Moncada, S. and Vane, J.R.: Prostacyclin release induced by bradykinin may contribute to the antihypertensive action of angiotensin-converting enzyme inhibitors. Advances in Prostaglandin and Throm-

boxane Research, 18.

Raven Press,

New York 7,

1159-1161,

1980.

Andersson, R.G.G., Saldeen, K. and Saldeen, T.: A fibrin(ogen) derived pentapeptide induces vasodilation, prostacyclin release and an increase in cyclic AMP.ThrombosisResearch. In press.