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Effect of different amounts of arachidonic acid on vessel wall-generated PGI2 and TXA2
Prostaglandins Leukotrienes and Medicine 12:
253 - 259, 1983
EFFECT OF DIFFERENT AMOUNTS OF ARACHIDONIC ACID ON VESSEL WALL-GENERATED PGI2 AND TXA2
Jawahar Mehta, Paulette Mehta, and Nancy Ostrowski From the Departments of Medicine and Pediatrics, University of Florida College of Medicine and the VA Medical Center, Gainesville, Florida.
ARSTRACT It has been suggested that low concentrations of AA may have vasoprotective and high concentrations vaso-damaging effects. To relate these effects to vascular generation of PGI2 and TXA2, we incubated human umbilical vein rings with AA (0, 0.01, 0.1, 1 and 2 mM) and examined the supernates for 6-keto-PGF and TXB . Low concentrations of AA (0.01 and 0.1 mM) caused prefere&al and Lima1 PGI relaase, whereas higher concentrations (1 and 2 mM) resulted in mark&l and preferential increase in TXA2 release. Disequilibrium in vascular PG12 and TXA release towards the latter may relate to vaso-damaging effects of h1gh concentrations of AA.
INTRODUCTION Since the discovery of PG12 and TXA2, these two derivatives of AA have been thought to play an important role in the regulation of vascular tone and integrity (1, 2). PG12 is believed to be generated primarily from the endothelial cells, and TXA2 mainly from the platelets. Several recent studies indicate that TX may also be synthesized by leukocytes (3) as well as from A2 animal blood vessels (4-6) and isolated endothelial cells (7). We have shown that human blood vessels are also capable of synthesizing TXA2 (8, 9). In addition, stimulation of human umbilical and other blood vessels with the substrate AA results in marked release of PG12 as well as TXA2 (8-10). In experimental animals (ll), infusion of small amounts of AA has been shown to be associated with vasodilator responses, and large amounts with sudden death (12). Ingerman-Wojenski et al (13) have demonstrated that infusion of AA in Key words:
AA - Arachidonic acid; PGI2 - Prostacyclin; TXA 253
2
- Thromboxane A . 2
high doses causes endothelial damage in rabbit arteries, whereas infusion of AA in low doses does not. These variable effects may relate to amounts of PGI2 and TKA2 synthesized by blood vessels in response to different amounts of AA. The present study was designed to examine relative PGI2/TKA generation by human blood vessels stimulated with variable concentrations oIAA.
MATERIALS ANDMETHODS
Preparation of Umbilical Veins: Umbilical cords were obtained after uncomplicated delivery. None of the mothers had taken prostaglandin-active drugs in the preceding 10 days. All experiments were completed within three hours of cord collection. The umbilical vein was gently dissected free from Wharton's jelly and cut into fine rings (0.5 to 1 mM thickness). Vascular rings (total wet weight 25-30 mg) were placed into small polypropylene vials containing 1 ml of Ca* and Mg*-free buffer. At the time of the experiment, the rings were gently washed several times with 1 ml of buffer and then placed in Hank's buffered salt solution (HBSS) containing Ca* and Mg* at 37' for 15 minutes in a water bath. The supernate was discarded. The rings were then incubated with HBSS alone or with AA 0.01, 0.1, 1 and 2 mM at 37OC for 15 minutes and the supernate collected. In each set of the experiments, five different aliquots of vascular rings from the same umbilical vein were prepared; one for HBSS alone and others for different concentrations of AA. Supernates (0,5 ml) were collected in 0.125 ml of aspirin (1 mM) and EDTA (4.5 mM) solution to prevent further in vitro degradation of AA. TKB2 was measured as a stable metabolite of TKA2 by Lyophilized TXB standards, 3H TKB2 and TXB2 antibody were obtained from New England Nutzear, Boston, MA. TKB2 levels in the test samples were obtained by comparison with TKB2 standards. Using this methodology, the cross-reactivity with other prostaglandins was as follows: 0.2% for PGE2, and less than 0.2% for PGA2, PGF2,, and 6-keto-PGFl,. All measurements were made in duplicate and results expressed in pg/mg tissue. Determination: 6-keto-PGFl, was measured as a stable hydrolysis 6-keto-PGF product ofLpG12 by radioimmunoassay using 6-keto-PGFlc standards, 3H 6-ketoPGFlo and antiserum to 6-keto-PGF u obtained from New England Nuclear, Boston, MA. Methodology was the same as ior TKB2 determination (15). Cross-reactivity with other prostaglandins was: PGF2o 2.7%, PGE2 2%, TKB2 < O.l%, PGA2 O.l%, PGAl < 0.3%. All measurements were made in duplicate and the results expressed in pglmg tissue. Confirmation of TXB2: TKB2 was confirmed in several ways. In some experiments, supernates were diluted serially (l:l, 1:2, 1:3, 1:4) and TKB2 antibody displacement compared to that of the known standard. In other experiments, the vascular rings were pretreated withcyclo-oxygenase inhibitor (aspirin, 1mM) or selective thromboxane synthesis inhibitor (OKY 1581, 0.1 mM) (16). After pretreatment with these agents, the vascular rings were stimulated with AA and the supernate examined for 6-keto-PGFlo and TKB2.
254
+t and Mg*-free buffer consisted of 138 mM NaCl, 4 mM KCl, Resgents: Ca 0.5 mk$Na2HPG4, 0.15 mu KH~PO~, 1.1 mM glucose, final pH 7.4. HBSS contained 1.3 mM CaC12, 2 H20, 5.4 mM KCl, 0.4 mM KH2PO4, 0.8 mM MgSO4.7 H20, 136.9 mM NaCl, 4.2 mM NaHCO3,0.3 mM Na2HP04, 5.6 mM dextrose adjusted to pH7.4 with 1 N NaOH. Aspirin (Sigma Chemical Co., St. Louis, MO) was dissolved in distilled water with gentle boiling. OKY 1581 (Ono Pharmaceutical Co., Osaka, Japan) was kept at -7OOC until use and dissolved in normal saline just prior to use. AA (Sigma Chemical Co., St. Louis, MO) was kept at -7OOC until use, and dissolved in tris buffer just prior to use (final pH 7.4). Calculations: Average of duplicate values was used for calculations of mean+SRM. Student's t-test was used for statistical analysis. A P-value less than 0.05 was considered significant.
RESULTS Spontaneous PG12 and TKA Release In all supernates -I 0 umbilical vein rings, both 6-keto-PGFl, and TKB2 were identified. The concentration of 6-keto-PGFlo ranged from 353 to 635 pg/mg (mean 468 2 86 pg/mg). The duplicate values varied by 15%. Different vascular rings from the same umbilical vein released similar amounts of PG12 (mean variation 11%). The concentration of TKB in the supernates ranged from 9 to 34 pg/mg (mean 212 4 pg/mg). The dupzicate TKR2 values were again similar (2 12%). Effect of AA on PGI and TKA2 Release (Fig. 1) With the lowesg concentration of AA (0.01 mM), 6-keto-PGF,o increased by 134% to 1110 t 112 pg/mg (P < 0.001). In contrast, TXR2 concentration was unchanged at 25 f. 4 pg/ml. With 0.1 mM AA the concentration of 6-keto-PGF was 284% higher compared to the control value (1796 + 161 pg/mg, P < O.OOl)'&d also to that with AA 0.01 mM (P < 0.001). TKB concentrations increased 200% to 63 If:14 pg/mg (P < 0.01 compared to controlf . With 1 mM AA, 6-keto-PGF was essentially unchanged at 1972 f. 160 pg/mg compared to 0.1 mM AA (P-N!!& In contrast, TKR concentrations increased dramatically by 762% to 181 + 20 pg/mg (P < O.OOf compared to baseline or AA 0.01 and 0.1 mM). With? mM kA, 6-keto-PGFl, concentrations did not show significant increase (mean 1976 + 164 pg/ml, P-NS compared to 0.1 or 1.0 mM AA). On the other hand, TKB2 concentrations showed a further increase to 214 t 20 pg/mg (P < 0.05 compared to 1 mM AA).
255
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160
.1600
120
1200
0 I 0 -3 c 2 800 f
IJo
400
40
0
L
0
0.01
0.1
0
1.0 2.0 mM AA
FIG. 1 Effect of AA on vascular PGI2 and TXA2 generation. Low concentrations of AA (0.01 and 0.1 mM) caused marked and maximal increase in PGI2, whereas high concentrations ( ) 0.1 mM) caused a preferential increase in T %'
6-keto-PGF /TXB Ratio (Fig. 2) this ratio was 22 + 4. With 0.01 mM AA, this ratio With &SFa$one, but increased to 49 k 7 (P < 0.01) indicating marked stimulation of PGI no change in TXA 0 With 0.1 mM AA this ratio decreased to 36 + 6, 8Lt with higher concentra8ions of AA, 6-keto-PGF /TXB ratio decreased-significantly indicating proportionately greater sti&Patioi of TXA2.
256
60
50
s
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EN
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30
k
3
P J
20
10
0 I
0
I
1 0.01 AAlrnMY1
I
I
1.0 2.0
FIG. 2 With low concentrations of AA (0.01 and 0.1 mM), PGI /TXA ratio ragio decreased. increased, whereas with high concentrations, PG12/d2
Confirmation of TXA2 in the Supernates a. Curves of TXR2 antibody displacement by TXR2 standards and by the supernates (undiluted and multiple dilutions) were similar suggesting similar kinetics (8). b.
Pre-treatment of vascular rings with aspirin resulted in 70 2 9% decrease in 6-keto-PGFle and 79 + 8% decrease in TXR2 concentration compared to other rings no pre-treated. Pre-treatment of the vascular rings with OKY 1581 followed by AA treatment resulted in 65 t 4% decreased TRR2 concentrations, but caused a lo-+ - 3% increase in 6-keto-PGFlo concentrations (8).
DISCUSSION This study confirms previous work showing that human umbilical veins in addition to PGI . Data from several experimental approaches indicate tha measured in tfe supernates was indeed TxB2. Relative TXA2 generation in response to substrate became in a wide range of concentrations, Lowest concentrations
257
i.e., 0.01 mM, caused a marked burst in PGI release, but no change in TXA2 release. At this concentration, the equl*jt ibrium between PGI and TXA2 was clearly in favor of the PGI2. At the next concentration of2AA, 0.1 mM, increase in PGI generation was still more than that in TXA2 (284% vs. ZOO%, P < 0.03). But the PG12 generation capability was maximal at 0.01 to 0.1 mM AA, since higher concentrations did not cause any significant additional increase. In contrast, TXA generation which was unaltered by the lowest concentration of AA, i.e., 0.61 mM, increased markedly with 0.1 mM AA. At higher concentrations of AA, > 0.1 mM, increase in TXA2 release was dramatic and persistent, such thzt with 1 or 2 mM AA, the balance between PG12 and TXA2 was clearly in favor of TXA2. used,
These differences in relative PG12 and TXA2 production in response to AA may relate to the observations that high concentrations may be vasoconstrictive and result in endothelial damage (13) and death (12). Marked increase in TXA may have systemic as well as localized detrimental effects (17). On the o$her hand, small concentrations of AA may have vasodilator effects by causing preferential PGI release which may be instrumental in preserving endothelial integrity (13, 18). Increased release of PGI may have protective effects related to its effects on Ca# flux and cycl1c AMP (19). Excessive amounts of TXA released in response to high concentrations of AA may have adverse effects gy lowering cyclic AMP. The significance of vessel wall-generated TXA is being increasingly recognized (20). These ex vivo studies indicate tZat a supply of substrate AA can influence amounts of PGI and TXA2 released by the vessel. These data on the synthesis of TXA2 and PGz2 by vessel walls may relate to the variable effects of AA upon its administration to the intact animal.
REFERENCES 1. MONCADA, S. and VANE, J.R. Arachidonic acid metabolites and the interactions between platelets and blood-vessel walls. N. Engl. J. Med. 300, 11421147, 1979. 2.
NEEDLEMAN, P. and KALEY, G. Cardiac and coronary prostaglandin synthesis and function. N. Engl. J. Med. 298, 122-128, 1978.
3.
WEKSLER, B.B. and GOLDSTEIN, I.M. Prostaglandins: Interactions with platelets and polymorphonuclear leukocytes in hemostasis and inflammation. Am. J. Med. 68, 419-428, 1980.
4.
MAIJRER,P., MOSKOVITZ, M.A., LEVINE, L. and MELAMED, E. The synthesis of prostaglandins by bovine cerebral microvessels. Prostaglandins Med. 5, 153-161, 1980.
5.
SALZMAN, P.M., SALMON, J.A. and MONCADA, S. Prostacyclin and thromboxane A2 synthesis by rabbit pulmonary artery. J. Pharmacol. Exp. Therap. 215, 240-247, 1980.
6.
HAGEN, A.A., WHITE, R.P. and ROBERTSON, J.T. Synthesis of prostaglandins and thromboxane B2 by cerebral arteries. Stroke 10, 306-309, 1979. 258
7.
INGERMAN-WOJENSKI, c., SILVER, M.J., SMITH, J.B. and MACARAK, E. Bovine endothelial cells in culture produce thromboxane as well as prostacyclin. J. Clin. Invest. 67, 1292-1296, 1981.
8.
MEHTA, P., MBHTA, J., CREWS, F,, ROY, L., OSTROWSKI, N. and HORALEK, C. Comparison of umbilical vein models for measurement of relative prostacyclin and thromboxane production. Prostaglandins 24, 743-750, 1982.
9.
MEHTA, J., MEHTA, P. and ROBERTS, A. Human vascular tissues produce thromboxane as well as prostacyclin. Am. J. Physiol. 1983 (in press).
10.
TWEMO, 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.
11.
ROSE, J.C., JOHNSON, M., RAMWELL, P.W,, et al. Effects of arachidonic acid on systemic arterial pressure, myocardial contractility and platelets in the dog. Proc. Sot. Exp. Biol. Med. 147, 652-659, 1974.
12.
SILVER, M.J., HOCH, W., KOCSIS, J.J., INGERMAN, C.M. and SMITH, J.B. Arachidonic acid causes sudden death in rabbits. Science 183, 1085-1087, 1974.
13.
INGERMAN-WOJENSKI, C., SILVER, M.J., SMITH, J.B., NISSENBADM, M. and SEDAR, A.W. Prostacyclin production in rabbit arteries in situ: Inhibition by arachidonic acid-induced endothelial cell damage or by low dose aspirin. Prostaglandins 21, 655-666, 1981.
14.
MEHTA, J. and MEHTA, P. Effects of propranolol therapy on platelet release and prostaglandin generation in patients with coronary heart disease. Circulation 66, 1294-1299, 1982,
15.
MEHTA, J, and MEHTA, P. Dipyridamole and aspirin in relation to platelet aggregation and vessel wall prostaglandin generation. J. Cardiovasc. Pharmacol. 4, 688-693, 1982.
16.
SMITH, J.B. and JUBIZ, W: OKY 1581: A selective inhibitor of thromboxane synthesis in vivo and in vitro. Prostaglandins 22, 353-362, 1981.
17.
SMITH, E.F., LEFER, A.M., AHARONY, D., SMITH, J.B., MAGOLDA, R.L., CLAREMON, D. and NICOLAOU, K.C. Carbocylic thromboxane A : Aggrevation of myocardial ischemia by a new synthetic thromboxane A2 ana1og. Prostaglandins 21, 443-455, 1981.
18.
LEFER, A.M., OGLETREE, MOL., SMITH, J.B., SILVER, M.J., NICOLAOU, K-C.,, BARNETTE, W.E. and GASIC, G.P. Prostacyclin: a potentially valuable agent for preserving myocardial tissue in acute myocardial ischemia. Science 200, 52-54, 1978.
19.
KUKOVETZ, W.R., HOLZMANN, S., WURM, A. and POCH, G. Prostacyclin increases CAMP in coronary arteries. J. Cyclic Nucleotide Res. 5, 469-476, 1979.
20.
ALLY, A.I. and HORROBIN, D,F. Thromboxane A in blood vessel walls and its physiological significance: Relevance to tfrombosis and hypertension. Prostaglandins Med. 4, 431-438, 1980.
PROST-
B
253 - 259, 1983
EFFECT OF DIFFERENT AMOUNTS OF ARACHIDONIC ACID ON VESSEL WALL-GENERATED PGI2 AND TXA2
Jawahar Mehta, Paulette Mehta, and Nancy Ostrowski From the Departments of Medicine and Pediatrics, University of Florida College of Medicine and the VA Medical Center, Gainesville, Florida.
ARSTRACT It has been suggested that low concentrations of AA may have vasoprotective and high concentrations vaso-damaging effects. To relate these effects to vascular generation of PGI2 and TXA2, we incubated human umbilical vein rings with AA (0, 0.01, 0.1, 1 and 2 mM) and examined the supernates for 6-keto-PGF and TXB . Low concentrations of AA (0.01 and 0.1 mM) caused prefere&al and Lima1 PGI relaase, whereas higher concentrations (1 and 2 mM) resulted in mark&l and preferential increase in TXA2 release. Disequilibrium in vascular PG12 and TXA release towards the latter may relate to vaso-damaging effects of h1gh concentrations of AA.
INTRODUCTION Since the discovery of PG12 and TXA2, these two derivatives of AA have been thought to play an important role in the regulation of vascular tone and integrity (1, 2). PG12 is believed to be generated primarily from the endothelial cells, and TXA2 mainly from the platelets. Several recent studies indicate that TX may also be synthesized by leukocytes (3) as well as from A2 animal blood vessels (4-6) and isolated endothelial cells (7). We have shown that human blood vessels are also capable of synthesizing TXA2 (8, 9). In addition, stimulation of human umbilical and other blood vessels with the substrate AA results in marked release of PG12 as well as TXA2 (8-10). In experimental animals (ll), infusion of small amounts of AA has been shown to be associated with vasodilator responses, and large amounts with sudden death (12). Ingerman-Wojenski et al (13) have demonstrated that infusion of AA in Key words:
AA - Arachidonic acid; PGI2 - Prostacyclin; TXA 253
2
- Thromboxane A . 2
high doses causes endothelial damage in rabbit arteries, whereas infusion of AA in low doses does not. These variable effects may relate to amounts of PGI2 and TKA2 synthesized by blood vessels in response to different amounts of AA. The present study was designed to examine relative PGI2/TKA generation by human blood vessels stimulated with variable concentrations oIAA.
MATERIALS ANDMETHODS
Preparation of Umbilical Veins: Umbilical cords were obtained after uncomplicated delivery. None of the mothers had taken prostaglandin-active drugs in the preceding 10 days. All experiments were completed within three hours of cord collection. The umbilical vein was gently dissected free from Wharton's jelly and cut into fine rings (0.5 to 1 mM thickness). Vascular rings (total wet weight 25-30 mg) were placed into small polypropylene vials containing 1 ml of Ca* and Mg*-free buffer. At the time of the experiment, the rings were gently washed several times with 1 ml of buffer and then placed in Hank's buffered salt solution (HBSS) containing Ca* and Mg* at 37' for 15 minutes in a water bath. The supernate was discarded. The rings were then incubated with HBSS alone or with AA 0.01, 0.1, 1 and 2 mM at 37OC for 15 minutes and the supernate collected. In each set of the experiments, five different aliquots of vascular rings from the same umbilical vein were prepared; one for HBSS alone and others for different concentrations of AA. Supernates (0,5 ml) were collected in 0.125 ml of aspirin (1 mM) and EDTA (4.5 mM) solution to prevent further in vitro degradation of AA. TKB2 was measured as a stable metabolite of TKA2 by Lyophilized TXB standards, 3H TKB2 and TXB2 antibody were obtained from New England Nutzear, Boston, MA. TKB2 levels in the test samples were obtained by comparison with TKB2 standards. Using this methodology, the cross-reactivity with other prostaglandins was as follows: 0.2% for PGE2, and less than 0.2% for PGA2, PGF2,, and 6-keto-PGFl,. All measurements were made in duplicate and results expressed in pg/mg tissue. Determination: 6-keto-PGFl, was measured as a stable hydrolysis 6-keto-PGF product ofLpG12 by radioimmunoassay using 6-keto-PGFlc standards, 3H 6-ketoPGFlo and antiserum to 6-keto-PGF u obtained from New England Nuclear, Boston, MA. Methodology was the same as ior TKB2 determination (15). Cross-reactivity with other prostaglandins was: PGF2o 2.7%, PGE2 2%, TKB2 < O.l%, PGA2 O.l%, PGAl < 0.3%. All measurements were made in duplicate and the results expressed in pglmg tissue. Confirmation of TXB2: TKB2 was confirmed in several ways. In some experiments, supernates were diluted serially (l:l, 1:2, 1:3, 1:4) and TKB2 antibody displacement compared to that of the known standard. In other experiments, the vascular rings were pretreated withcyclo-oxygenase inhibitor (aspirin, 1mM) or selective thromboxane synthesis inhibitor (OKY 1581, 0.1 mM) (16). After pretreatment with these agents, the vascular rings were stimulated with AA and the supernate examined for 6-keto-PGFlo and TKB2.
254
+t and Mg*-free buffer consisted of 138 mM NaCl, 4 mM KCl, Resgents: Ca 0.5 mk$Na2HPG4, 0.15 mu KH~PO~, 1.1 mM glucose, final pH 7.4. HBSS contained 1.3 mM CaC12, 2 H20, 5.4 mM KCl, 0.4 mM KH2PO4, 0.8 mM MgSO4.7 H20, 136.9 mM NaCl, 4.2 mM NaHCO3,0.3 mM Na2HP04, 5.6 mM dextrose adjusted to pH7.4 with 1 N NaOH. Aspirin (Sigma Chemical Co., St. Louis, MO) was dissolved in distilled water with gentle boiling. OKY 1581 (Ono Pharmaceutical Co., Osaka, Japan) was kept at -7OOC until use and dissolved in normal saline just prior to use. AA (Sigma Chemical Co., St. Louis, MO) was kept at -7OOC until use, and dissolved in tris buffer just prior to use (final pH 7.4). Calculations: Average of duplicate values was used for calculations of mean+SRM. Student's t-test was used for statistical analysis. A P-value less than 0.05 was considered significant.
RESULTS Spontaneous PG12 and TKA Release In all supernates -I 0 umbilical vein rings, both 6-keto-PGFl, and TKB2 were identified. The concentration of 6-keto-PGFlo ranged from 353 to 635 pg/mg (mean 468 2 86 pg/mg). The duplicate values varied by 15%. Different vascular rings from the same umbilical vein released similar amounts of PG12 (mean variation 11%). The concentration of TKB in the supernates ranged from 9 to 34 pg/mg (mean 212 4 pg/mg). The dupzicate TKR2 values were again similar (2 12%). Effect of AA on PGI and TKA2 Release (Fig. 1) With the lowesg concentration of AA (0.01 mM), 6-keto-PGF,o increased by 134% to 1110 t 112 pg/mg (P < 0.001). In contrast, TXR2 concentration was unchanged at 25 f. 4 pg/ml. With 0.1 mM AA the concentration of 6-keto-PGF was 284% higher compared to the control value (1796 + 161 pg/mg, P < O.OOl)'&d also to that with AA 0.01 mM (P < 0.001). TKB concentrations increased 200% to 63 If:14 pg/mg (P < 0.01 compared to controlf . With 1 mM AA, 6-keto-PGF was essentially unchanged at 1972 f. 160 pg/mg compared to 0.1 mM AA (P-N!!& In contrast, TKR concentrations increased dramatically by 762% to 181 + 20 pg/mg (P < O.OOf compared to baseline or AA 0.01 and 0.1 mM). With? mM kA, 6-keto-PGFl, concentrations did not show significant increase (mean 1976 + 164 pg/ml, P-NS compared to 0.1 or 1.0 mM AA). On the other hand, TKB2 concentrations showed a further increase to 214 t 20 pg/mg (P < 0.05 compared to 1 mM AA).
255
.2400
200
'2000
160
.1600
120
1200
0 I 0 -3 c 2 800 f
IJo
400
40
0
L
0
0.01
0.1
0
1.0 2.0 mM AA
FIG. 1 Effect of AA on vascular PGI2 and TXA2 generation. Low concentrations of AA (0.01 and 0.1 mM) caused marked and maximal increase in PGI2, whereas high concentrations ( ) 0.1 mM) caused a preferential increase in T %'
6-keto-PGF /TXB Ratio (Fig. 2) this ratio was 22 + 4. With 0.01 mM AA, this ratio With &SFa$one, but increased to 49 k 7 (P < 0.01) indicating marked stimulation of PGI no change in TXA 0 With 0.1 mM AA this ratio decreased to 36 + 6, 8Lt with higher concentra8ions of AA, 6-keto-PGF /TXB ratio decreased-significantly indicating proportionately greater sti&Patioi of TXA2.
256
60
50
s
40
3
EN
‘-
30
k
3
P J
20
10
0 I
0
I
1 0.01 AAlrnMY1
I
I
1.0 2.0
FIG. 2 With low concentrations of AA (0.01 and 0.1 mM), PGI /TXA ratio ragio decreased. increased, whereas with high concentrations, PG12/d2
Confirmation of TXA2 in the Supernates a. Curves of TXR2 antibody displacement by TXR2 standards and by the supernates (undiluted and multiple dilutions) were similar suggesting similar kinetics (8). b.
Pre-treatment of vascular rings with aspirin resulted in 70 2 9% decrease in 6-keto-PGFle and 79 + 8% decrease in TXR2 concentration compared to other rings no pre-treated. Pre-treatment of the vascular rings with OKY 1581 followed by AA treatment resulted in 65 t 4% decreased TRR2 concentrations, but caused a lo-+ - 3% increase in 6-keto-PGFlo concentrations (8).
DISCUSSION This study confirms previous work showing that human umbilical veins in addition to PGI . Data from several experimental approaches indicate tha measured in tfe supernates was indeed TxB2. Relative TXA2 generation in response to substrate became in a wide range of concentrations, Lowest concentrations
257
i.e., 0.01 mM, caused a marked burst in PGI release, but no change in TXA2 release. At this concentration, the equl*jt ibrium between PGI and TXA2 was clearly in favor of the PGI2. At the next concentration of2AA, 0.1 mM, increase in PGI generation was still more than that in TXA2 (284% vs. ZOO%, P < 0.03). But the PG12 generation capability was maximal at 0.01 to 0.1 mM AA, since higher concentrations did not cause any significant additional increase. In contrast, TXA generation which was unaltered by the lowest concentration of AA, i.e., 0.61 mM, increased markedly with 0.1 mM AA. At higher concentrations of AA, > 0.1 mM, increase in TXA2 release was dramatic and persistent, such thzt with 1 or 2 mM AA, the balance between PG12 and TXA2 was clearly in favor of TXA2. used,
These differences in relative PG12 and TXA2 production in response to AA may relate to the observations that high concentrations may be vasoconstrictive and result in endothelial damage (13) and death (12). Marked increase in TXA may have systemic as well as localized detrimental effects (17). On the o$her hand, small concentrations of AA may have vasodilator effects by causing preferential PGI release which may be instrumental in preserving endothelial integrity (13, 18). Increased release of PGI may have protective effects related to its effects on Ca# flux and cycl1c AMP (19). Excessive amounts of TXA released in response to high concentrations of AA may have adverse effects gy lowering cyclic AMP. The significance of vessel wall-generated TXA is being increasingly recognized (20). These ex vivo studies indicate tZat a supply of substrate AA can influence amounts of PGI and TXA2 released by the vessel. These data on the synthesis of TXA2 and PGz2 by vessel walls may relate to the variable effects of AA upon its administration to the intact animal.
REFERENCES 1. MONCADA, S. and VANE, J.R. Arachidonic acid metabolites and the interactions between platelets and blood-vessel walls. N. Engl. J. Med. 300, 11421147, 1979. 2.
NEEDLEMAN, P. and KALEY, G. Cardiac and coronary prostaglandin synthesis and function. N. Engl. J. Med. 298, 122-128, 1978.
3.
WEKSLER, B.B. and GOLDSTEIN, I.M. Prostaglandins: Interactions with platelets and polymorphonuclear leukocytes in hemostasis and inflammation. Am. J. Med. 68, 419-428, 1980.
4.
MAIJRER,P., MOSKOVITZ, M.A., LEVINE, L. and MELAMED, E. The synthesis of prostaglandins by bovine cerebral microvessels. Prostaglandins Med. 5, 153-161, 1980.
5.
SALZMAN, P.M., SALMON, J.A. and MONCADA, S. Prostacyclin and thromboxane A2 synthesis by rabbit pulmonary artery. J. Pharmacol. Exp. Therap. 215, 240-247, 1980.
6.
HAGEN, A.A., WHITE, R.P. and ROBERTSON, J.T. Synthesis of prostaglandins and thromboxane B2 by cerebral arteries. Stroke 10, 306-309, 1979. 258
7.
INGERMAN-WOJENSKI, c., SILVER, M.J., SMITH, J.B. and MACARAK, E. Bovine endothelial cells in culture produce thromboxane as well as prostacyclin. J. Clin. Invest. 67, 1292-1296, 1981.
8.
MEHTA, P., MBHTA, J., CREWS, F,, ROY, L., OSTROWSKI, N. and HORALEK, C. Comparison of umbilical vein models for measurement of relative prostacyclin and thromboxane production. Prostaglandins 24, 743-750, 1982.
9.
MEHTA, J., MEHTA, P. and ROBERTS, A. Human vascular tissues produce thromboxane as well as prostacyclin. Am. J. Physiol. 1983 (in press).
10.
TWEMO, 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.
11.
ROSE, J.C., JOHNSON, M., RAMWELL, P.W,, et al. Effects of arachidonic acid on systemic arterial pressure, myocardial contractility and platelets in the dog. Proc. Sot. Exp. Biol. Med. 147, 652-659, 1974.
12.
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PROST-
B