Myocardial prostacyclin and thromboxane A2 synthetase activities during ischemia and reperfusion in isolated rabbit heart

The aim of the study was to determine the prostacyclin (PGI2) and thromboxane A2 (TXA2) synthetase activities of myocardial tissue and their variation during ischemia and reperfusion. Regional ischemia was induced by 10 min occlusion of the left anterior descending coronary artery in isolated Langendorff rabbit hearts. Biosynthesis of PGI2 and TXA2 were carried out by using arachidonic acid as substrate and left ventricle microsomes (LVM) from ischemic and non-ischemic areas as sources of PGI2 and TXA2 synthetase. 6-keto-PGF1 alpha and TXB2, stable metabolites of PGI2 and TXA2 respectively, were determined by radioimmunoassay. Experiments carried out under the adopted conditions showed that LVM were able to synthetise PGI2 as well as TXA2 from arachidonic acid. On the other hand, ischemia depressed both PGI2 and TXA2 synthetase activities of cardiac tissue: the depression was more pronounced on TXA2 synthetase than on PGI2 synthetase with no significant difference between ischemic and non-ischemic regions. Moreover, ischemia increased the ratio 6-keto-PGF1 alpha/TXB2 indicating therefore that it can facilitate the formation of PGI2. The post ischemic reperfusion of the heart counteracted the decrease in PGI2 synthetase induced by ischemia which returned to the normal level: reperfusion also slightly reversed the decrease in TXA2 the decrease in TXA2 synthetase. However, the diminution in TXA2 synthetase of non-ischemic myocardium was attenuated but it remained lower than the normal level. These results suggested that the whole left ventricle is affected by regional ischemia. Furthermore it appears that myocardial TXA2 synthetase is more vulnerable than PGI2 synthetase to a lack of oxygen and nutrients.     

Carbocyclic thromboxane A2: Aggrevation of myocardial ischemia by a new synthetic thromboxane A2 analog

$\text{In vitro}$ experiments indicate that thromboxane A₂ (TA₂) is a potent platelet aggregator and vascular constrictor. However, it is unclear what roles these specific actions may contribute in the pathophysiology of myocardial ischemia. Carbocyclic thromboxane A₂ (CTA₂), a TA₂ analog, constricts isolated perfused cat coronary arteries, but does not aggregate platelets, and thus appeared useful to clarify these separate actions of TA₂. In anesthetized cats, radioactive labeled microspheres were injected into the left atrium for measurement of cardiac output and tissue blood flows. Compared to control measurements, CTA₂ infusion (4.8 μg·kg^?1·min^?1 to 10 min) significantly decreased cardiac output from 347 ± 16 ml·min^?1 to 248 ± 16 ml·min^?1 (p<0.025). Furthermore, V7 CTA₂ also significantly reduced blood flow to the left ventricle by 33 ± 7%, but did not alter heart rate or MABP in the intact cat. In cats subjected to left anterior descending coronary artery occlusion, infusion of CTA₂ (1 μg·min^?1 for 120 minutes) 30 min after ligation resulted in a significantly reduced myocardial cellular integrity as measured by myocardial creatine kinase activity (p<0.01) or percent bound myocardial cathepsin D (p<0.01). Thus, these data suggest that activation of vascular thromboxane receptors as well as direct cellular damage may play a role in the pathophysiology of myocardial ischemia.     

Respiratory movements alter the generation of prostacyclin and thromboxane A2 in isolated rat lungs: The influence of arachidonic acid-pathway inhibitors on the ratio between pulmonary prostacyclin and thromboxane A2

The influence of hyperventilation on the spontaneous generation of prostacyclin and thromboxane A₂ by isolated rat lungs was studied. Both prostacyclin and thromboxane A₂, as measured by RIA of their stable end-products, 6-oxo-PGF_1α and TXB₂ respectively, were continuously released into the perfusate. However, the concentration of prostacyclin in the perfusate was higher than thromboxane A₂. Under normal ventilation at a rate 40–50 breaths/min, the ratio between these two compounds was 5:1. Increasing the rate of respiration to 100 breaths/min preferentially stimulated the release of prostacyclin. During hyperventilation, the ratio between 6-oxo-PGF_1α and TXB₂ was 12:1. Aspirin and indomethacin suppressed both basal and hyperventilation-stimulated release of prostacyclin and thromboxane A₂. Hydroperoxy-fatty acids and tranylcypromine inhibited only the release of prostacyclin but did not affect the generation of thromboxane A₂. Our findings confirm that the lung generates prostacyclin predominantly, and provide direct evidence that respiratory movements are involved in generation of pulmonary prostacyclin and thromboxane A₂.     

Prostacyclin and thromboxane A2 release in isolated rat lungs

The influence of platelets and platelet membranes on the generation of prostacyclin (PGI₂) and thromboxane A₂(TXA₂) by isolated rat lung and porcine aortic endothelial cell, as measured by RIA of their stable end-producs, 6-oxo-PGF_1α and TXB₂ respectively, was studied. After introduction of either aspirin-treated platelets or membranes from aspirin-treated platelets to the perfusate, 1 5-fold increase in the amount of 6-oxo-PGF_1α and TXB₂ in the perfusate was observed. Treatment of the lung with aspirin produced a 50% reduction in the platelet-stimulated release of PGI₂ and TXA₂. Treatment of the lung with the phospholipase inhibitor, mepacrine, significantly reduced the platelet-stimulated release of PGI₂ and TXA₂. Incubation of endothelial cells with untreated platelet membranes did not alter the generation of PGI₂. These results suggest that platelet-stimulated release of PGI₂ and TXA₂ occurs via mechanical stimulation of phospholipase A₂, liberating arachidonic acid.     

Modulation of platelet thromboxane A2 and arterial prostacyclin by dietary vitamin E

Platelets from vitamin E-deficient and vitamin E-supplemented rats generate the same amount fo thromboxane A₂ (TxA₂) when they are incubated with unesterified arachidonic acid. Platelets from vitamin E-deficient rats produce more TxA₂ than platelets from vitamin E-supplemented rats when the platelets are challenged with collagen. Arterial tissue from vitamin E-deficient rats generates less prostacyclin (PGI₂) than arterial tissue from vitamin E-supplemented rats. The vitamin E effect with arterial tissue is observed when the tissue is incubated with and without added unesterified arachidonic acid. These data show that arterial prostacyclin synthesis is diminished in vitamin E-deficient rats. Vitamin E, $\text{in}$$\text{vivo}$, inhibits platelet aggregation both by lowering platelet TxA₂ and by raising arterial PGI₂.     

Increased thromboxane A2 production but normal prostacyclin by the placenta in hypertensive pregnancies

The production of vasodilatory, antiaggregatory prostacyclin (PGI₂) and vasoconstrictory, proaggregatory thromboxane A₂ (TxA₂) by the placenta was studied in the cases of hypertensive pregnancy complications by superfusing pieces from maternal and fetal sides of placentae of 9 pre-eclamptic, 6 hypertensive and 11 healthy women and measuring the release of 6-keto-prostaglandin F_1α (6-keto-PGF_1α) and thromboxane B₂ (TxB₂), the breakdown products of PGI₂ and TxA₂ respectively, from the superfusate. Both sides of the placentae from the controls produced 6-keto-PGF_1α (maternal side 0.5±0.1 ng/g/min dry weight of tissue, mean±SEM; fetal side 0.7±0.2 ng/g/min) and TxB₂ (maternal side 2.5±0.4 ng/g/min; fetal side 2.7±0.5 ng/g/min with no correlation between the two. The 6-keto-PGF_1α production was normal in hypertensive complications whereas the TxB₂ production was increased on the fetal side of the placentae obtained from the pre-eclamptic (3.7±0.3 ng/g/min: p<0.05) and hypertensive women (4.1±0.4 ng/g/min; p<0.025). This may explain the occurrence of microthrombi and infarctions in placentae of hypertensive women.     

Alterations of vascular prostacyclin and thromboxane A2 in Dahl genetical strain susceptible to salt-induced hypertension

To assess the implications of vascular eicosanoids system in the hypertension of Dahl salt-sensitive (Dahl S) strain, we investigated the production of vascular vasodepressor and vasoconstrictor eicosanoids in Dahl S rats. 14-week-old Dahl S rats on a 0.11% NaCl diet (normotension) or a 0.3% NaCl diet (borderline hypertension) had a significantly lowered generation of vascular prostacyclin (PGI₂), compared with Dahl salt-resistant (Dahl R) rats. The impairment of vascular PGI₂ in Dahl S rats was restored to the normal level of Dahl R rats with the elevation of blood pressure induced by a high salt diet (4% NaCl). The production of vascular PGI₂ was closely related to the height of blood pressure. The deterioration of vascular PGI₂ was also found in 4-week-old Dahl S rats with normotension. Conversely, vascular thromboxane A₂ (TXA₂) was significantly enhanced in 14-week-old Dahl S rats in all of the feeding groups. Thus, it seems possible that the proved alterations of the vasodepressor and vasoconstrictor eicosanoids partially contribute to the genesis of salt hypertension. Although the exact mechanisms remain obscure, the adaptation of vascular PGI₂ on a high salt diet may be suitable to compete with the high blood pressure and to protect against the vascular damage.     

The anti-thromboxane A2 synthetase activity of myocardial tissue and its variation during ischemia and reperfusion in isolated rabbit heart.

In this investigation, an anti-thromboxane A2 (TXA2) synthetase activity in the myocardial tissue, which can be modulated by ischemia and reperfusion, was observed. Regional ischemia was induced by 60 min occlusion of the left anterior descending coronary artery in isolated Langendorff rabbit hearts. Biosynthesis of TXA2 was carried out by using arachidonic acid (AA) as substrate, horse platelet microsomes (HPM) as the source of TXA2 synthetase and left ventricle microsomes (LVM) from ischemic and non-ischemic areas as effectors TXB2, the stable metabolite of TXA2, was determined by radioimmunoassay. Experiments carried out under the adopted conditions showed that LVM from control hearts were able to inhibit by up to 50% the biosynthesis of TXA2 from HPM. This anti-TXA2 synthetase activity was more pronounced when LVM from the non-ischemic area were used, rather then LVM from the ischemic one. A 60 min reperfusion decreased the anti-TXA2 activity. A superfused rabbit aorta strip was also used as a cascade bioassay to study the effect of LVM on the TX2-synthetase activity of HPM, and this confirmed our findings. These results suggest that the left ventricle possesses a self-defense mechanism against acute myocardial ischemia, independently from the circulation. The postulated mechanism may be initiated in the non-ischemic area.     

cAMP dependent inhibition of thromboxane A2, prostacyclin and PGF2α synthesis in mouse hepatocytes

The role of cAMP dependent regulation in thromboxane A₂, prostacyclin and PGF_2α synthesis (measured by radioimmunoassay) was investigated in isolated mouse hepatocytes and in microsomal membranes prepared from these cells. In isolated hepatocytes N⁶,O²-dibutyryl cAMP inhibited the formation of all the three derivatives, while calcium ionophore A 23187 stimulated their synthesis. Addition of the dissociated catalytic subunit of cAMP dependent protein kinase and ATP to microsomal membranes inhibited the production of TXA₂, PGI₂ and PGF_2α by about 50% and this inhibition was counteracted by the combined addition of heat stable inhibitor protein of cAMP dependent protein kinase. It is concluded that in parenchylmal liver cells cAMP dependent phosphorylation is directly involved in the inhibition of prostanoid synthesis.     

Experimental atherosclerosis in rabbits: Platelet aggregation, thromboxane A2 generation and anti-aggregatory potency of prostacyclin

Experimental atherosclerosis in rabbits was associated with increased aggregation of their platelets to arachidonic acid, and with increased generation of thromboxane A₂ by their platelet-rich plasma. A heightened susceptibility of platelets to the anti-aggregatory action of prostacyclin against the ADP-induced aggregation was also observed. It is concluded that in advanced atherosclerosis the platelet system is hypersensitive to biologically active metabolites of arachidonic acid.     

Plasma endotoxin and concentrations of stable metabolites of prostacyclin, thromboxane A2, and prostaglandin E2 in postpartum dairy cows

The presence of endotoxin in plasma and patterns of stable metabolites of prostacyclin (PC), thromoxane A₂ (TXA₂) and prostaglandin E₂ (PGE₂) were determined during the first postpartum estrous cycles in sixteen dairy cows. These included 8 cows with uterine infections which exhibited shortened luteal phases (SC) and 8 cows which had normal luteal phases (NC) after the first post partum ovulations. Endotoxin was consistently detected in all SC cows during the abbreviated estrous cycles while plasma samples of NC cows were free of endotoxin. Plasma concentrations of TXA₂ metabolite was higher in SC cows (p<0.05) (1785–3452 pg/ml) compared to NC cows (723–1240 pg/ml). Similarly, plasma concentrations of PC metabolite was higher in SC cows (p<0.07) (423–1847 pg/ml) compared to NC cows (159–325 pg/ml). In contrast, plasma concentrations of PGE₂ metabolite was higher in NC cows (p<0.05) (850–2219 pg/ml) compared to SC cows (455–628 pg/ml). The results of this study suggest that postpartum uterine infections mediate the release of prostaglandins from the uteri by means of the endotoxin and endotoxin appears to stimulate selectively the production of PC and TXA₂ favoring early demise of corpora lutea formed after first postpartum ovulations in dairy cows.     

Effects of a thromboxane synthetase inhibitor and a thromboaxane antagonist on release and activity of thromboxane A2 and prostaglandin in vitro

The TxA₂ synthetase inhibitor, dazoxiben, and the TxA₂ antagonist, ±SQ 29, 548, were examined for effects on release and vasoactivity of TxA₂ and prostacyclin. Isolated perfused guinea pig lungs were used as the enzyme source from which TxA₂ and prostacyclin were released in response to injections of arachidonic acid or bradykinin. Both dazoxiben and ±SQ 29, 548 inhibited contraction of the superfused rat aorta and bovine coronary artery after arachidonic acid injection through the lung. ±SQ 29, 548 abolished contractions of the rat aorta, but significant aorta contracting activity persisted during dazoxiben treatment. Dazoxiben significantly inhibited arachidonate-induced release of TxA₂ (immunoreactive TxB₂)iinto the superfusate, but TxA₂ release was significantly potentiated by ±SQ 29, 548. Thus, in the presence of enhanced TxA₂ concentrations, ±SQ 29, 548 effectively antagonized the vasospastic effect of TxA₂. Dazoxiben diverted a significantly greater amount of arachidonic acid into prostacyclin synthesis (immunoreactive 6-keto-PGF_1α), changing original coronary vasoconstriction into relaxation. ±SQ 29, 548 did not significantly modify lung prostacyclin synthesis. Moreover, with ±SQ 29, 548, the absence of TxA₂-mediated coronary contraction unmasked active relaxation of the superfused bovine coronary artery, coincident with thromboxane and prostacyclin release. Dazoxiben consistently inhibited TxA₂ synthesis and enhanced prostacyclin synthesis. ±SQ 29, 548 augmented TxB₂ release in response to arachidonate, but not bradykinin, and did not significantly alter 6-keto-PGF_1α release in response to either arachidonate or bradykinin. In terms of vasoactivity measured , ±SQ 29, 548 and dazoxiben produced similar anti-vasospastic effects, although this was accomplished by completely different mechanisms.     

Effects of nitrogen dioxide exposure on prostacyclin synthesis in lung and thromboxane A2 synthesis in platelets in rats

Effects of nitrogen dioxide (NO₂) exposure on prostacyclin (PGIP₂) synthesis in the rat lung and thromboxane A₂ (TXA₂) synthesis in the platelets were studied. Male Wistar rats were exposed to 10 ppm NO₂ for 1, 3, 5, 7 and 14 days. PGI₂ synthesizing activity of homogenized lung decreased. The damage of PGI₂ synthesizing activity reaches its maximum at 3 days. At 14 days, PGI₂ synthesizing activity returned to the normal level. The activity of PGI₂ synthetase decreased significantly. The formation of lipid peroxides due to NO₂ exposure may cause the depression of PGI₂ synthesizing activity of lung. On the other hand, platelet TXA₂ synthesizing activity increased. This increased TXA₂ synthesizing activity lasted at least till 3 days. Then, it returned to the normal level. The counts of platelet were decreased significantly by 1, 3, 5 and 7 days NO₂ exposure. Then the decreased counts of platelet returned to the normal level at 14 days NO₂ exposure. These results indicate that the depression of PGI₂ synthesizing activity lung by NO₂ exposure cause an increase in TXA₂ synthesizing activity of platelets. It may contribute to induce platelet aggregation and to the observed decrease in the number of platelets during NO₂ exposure.     

Production of thromboxane A2 by the kidney in glycerol-induced acute renal failure in the rabbit

The hemodynamic alterations occuring in glycerol induced renal failure are controversial. To date no single humoral substance can fully explain the change in renal resistance observed in this hemodynamic model of acute renal failure. To assess the capacity of the rabbit kidney to produce thromboxane A₂, a potent vasoconstrictor, the following experiments were carried out. Rabbits received 14 ml/kg of 50% glycerol subcutaneously 24 hrs before the study. After 24 hrs., the kidneys were removed and perfused ex vivo in superfusion bioassay cascade. Kidneys from rabbits which developed renal failure, as assessed by elevated serum creatinines, released a substance which produced contraction of rabbit aorta (RCS) in response to bradykinin (BK) and angiotensin II. Microsomes prepared from these kidneys when incubated with [¹⁴C]-arachidonic acid produced a peak of radioactivity which comigrated with thromboxane B₂ on thin layer chromatography and was inhibited by the thromboxane synthetase inhibitor imidazole. Furthermore, an inverse linear relation was found between the BK dose required to release RCS from perfused kidney and the serum creatinine levels. A direct linear relation was found between the percent of TxB₂ produced by renal microsome preparations and the serum creatinine.These studies demonstrate an increased renal capacity of the glycerol-model of acute renal failure to produce TxA₂. The production of TxA₂ a potent vasoconstrictor should therefore be further evaluated as a potential endogenous mediator of the hemodynamic changes occurring in acute renal failure.     

Platelet rich plasma transforms exogenous prostaglandin endoperoxide H2 into thromboxane A2

Platelet rich plasma transforms exogenous prostaglandin endoperoxide H₂ into thromboxane A₂ immediately prior to the initiation of irreversible aggregation. Selective thromboxane synthetase inhibitors block thromboxane A₂ formation and aggregation. Thromboxane A₂ formation appears to be essential during arachidonate mediated aggregation. The results presented reconcile the previously accepted paradoxical behavior of thromboxane synthetase in platelet rich plasma toward the prostaglandin endoperoxide H₂ substrate.     

Polymorphonuclear leukocytes produce thromboxane A2-like activity during phagocytosis

Homogenates of phagocytosing polymorphonuclear leukocytes obtained from rabbit peritoneum were incubated with the prostaglandin endoperoxides PGG₂ or PGH₂. After 2 min at 0°C, incubation mixtures contained an increased rabbit aorta contracting activity. Ether extracts of incubation mixtures contained a substance which contracted the superfused strips of rabbit aorta and coeliac artery and had a half life which was similar to thromboxane A₂. The generation of thromboxane A₂-like activity from PG endoperoxides was prevented by boiling the homogenate prior to incubation, or by pretreatment with benzydamine, a drug which blocks thromboxane formation in platelets. Production of thromboxane A₂-like material by leukocyte homogenates was compared with platelet microsomal thromboxane synthetase.     

Ultrastructural changes of sarcolemma and mitochondria in the isolated rabbit heart during ischemia and reperfusion

Isolated rabbit hearts were perfused by the Langendorff technique, made ischemic and subsequently reperfused. It was found that ischemia results in: (i) aggregation of the intramembranous particles in the sarcolemma and (ii) extrusion of pure lipidic multilamellar structures (liposomes) from swollen mitochondria. Subsequent reperfusion resulted in further aggregation of the sarcolemmal intramembranous particles and disruption of the sarcolemma, which was attended by the formation of liposome-like structures. Intramembrane particle aggregation is explained in terms of lateral phase separation of the membrane lipids and a reduction of repulsive forces between the membrane proteins, both induced by a decrease in pH and an increase in Ca2+ concentration intracellularly. The formation and extrusion of the multilamellar structures are discussed in terms of destabilization of the bilayer which results in a structural blebbing-off of pure lipid.     

Biological activities of new gibberellins A30-A35 and A35 glucoside

The plant growth-promoting activities of new gibberellins, GA₃₀, GA₃₀, GA₃₂, GA₃₃, GA₃₄, GA₃₅ and GA₃₅ glucoside were evaluated in seven bioassays. In general GA₃₀, GA₃₀, and GA₃₅ showed fairly high biological activities, whilst GA₃₃, GA₃₄ and GA₃₅ glucoside were almost inactive. GA₃₂ was highly active, behaving similarly to GA₃. It is suggested that the C-11β and C-12α hydroxyl groups have little influence on growth-promoting activity, although the C-12α hydroxyl group reduces activity in the cucumber hypocotyl assay.     

Antibodies against type II phospholipase A2 prevent renal injury due to ischemia and reperfusion in rats

This study was performed to determine the involvement of type II phospholipase A₂ (PLA₂-II) in renal injury caused by ischemia and reperfusion. Ischemia and reperfusion significantly elevated levels of blood urea nitrogen and serum creatinine in rats. These increases were significantly reduced by i.v. administration of rabbit IgG F(ab′)2 fragments against rat PLA₂-II. Increased levels of acid-stable PLA₂ activity in the kidney were caused by ischemia and reperfusion, and were suppressed by administration of anti-PLA₂-II F(ab′)2. Increased levels of myeloperoxidase activity, a marker of neutrophil infiltration, in the kidney were also reduced after anti-PLA₂-II F(ab′)2 treatment. These results suggest that PLA₂-II plays a pivotal role in pathogenesis of ischemia and reperfusion injury through induction of neutrophil infiltration.