The composition and physicochemical properties of the blood lipoproteins in rats exposed to external gamma irradiation]

Content of lipids, character of chemiluminescence of blood plasma and certain classes of lipoproteins have been studied. Geometrical parameters, nature and quantity of charged groups of lipoprotein particles accessible for titration have been determined 1 and 30 days after a single external gamma irradiation of rats in a dose of 3 Gy. The used irradiation dose exerts an expressed hyperlipidemic effect retained for one month after irradiation. The disturbances in the spectrum of blood lipids and lipoproteins are of hyper-beta and hyper-prebeta lipoproteinemia character. Considerable disturbances of physicochemical properties of different classes of lipoproteins have been detected. They are exhibited in changes of the pattern of free-radical processes, state of the charge of surface ionogenic groups and geometrical parameters of lipoprotein particles. Changes registered by the methods of potentiometric titration and correlation spectroscopy are most expressed in lipoproteins of very low density and those of low density.     

Acetyl glycerylphosphorylcholine inhibition of prostaglandin I2-stimulated adenosine 3',5'-cyclic monophosphate levels in human platelets. Evidence for thromboxane A2 dependence

Previous studies with AGEPC (1-O-hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) stress the independence of the proaggregatory activity of AGEPC from the platelet cyclooxygenase. However, our dose response analyses in human platelet-rich plasma show distinct primary and secondary waves of aggregation in response to AGEPC. Second wave aggregation is inhibited completely by either 10 micro M indomethacin, a cyclooxygenase inhibitor, or 5.6 micro M 9,11-azoprosta-5,13-dienoic acid, a thromboxane A2 synthetase inhibitor. Simultaneous addition of AGEPC and prostaglandin I2 to platelet-rich plasma results in a marked increase in platelet cyclic AMP, which is not different from the prostaglandin I2 response alone. However, if prostaglandin I2 is added to AGEPC-stimulated platelets at a point where secondary aggregation is just beginning, AGEPC can attenuate prostaglandin I2-stimulated cyclic AMP accumulation. The inhibition by AGEPC is blocked by either cyclooxygenase or thromboxane A2 synthetase inhibitors, and radioimmunoassay of thromboxane B2 confirmed that the inhibition of prostaglandin I2-stimulated cyclic AMP accumulation is due to thromboxane A2 synthesis, and that AGEPC-stimulated secondary aggregation does not start until thromboxane A2 is synthesized. These data suggest that much of the bioactivity of AGEPC is attributable to thromboxane A2.     

Plasma lipoproteins affect platelet malondialdehyde and thromboxane B2 production

Platelet interaction with plasma lipoproteins was studied using gel-filtered platelets free of plasma constituents and purified lipoproteins. On incubation of gel-filtered platelets with plasma lipoproteins at 30 degrees C for 30 min, 100 micrograms of protein/ml of very-low as well as low-density lipoprotein caused 10% increment in platelet aggregation and [14C]serotonin release in parallel to elevation of around 15% of malondialdehyde and thromboxane B2 production. High-density lipoprotein showed the opposite effect and reduced platelet aggregation as well as thromboxane B2 synthesis by 17 and 32%, respectively. Lipoprotein-deficient plasma enhanced platelet function. Preincubation of the platelet suspension with prostacyclin did not prevent the effect of the lipoproteins on the in vitro platelet response as well as on the platelet prostaglandin pathway. Our results suggest that the formation of thromboxane B2 and malondialdehyde is influenced by plasma lipoproteins and that these, in turn, affect platelet aggregation and the release reaction. The possible significance of these results to platelet function in hyperlipidemic patients is discussed.     

Urinary excretion of cyclic nucleotides, creatinine prostaglandin E2 and thromboxane B2 from mice exposed to whole-body irradiation from an enhanced neutron field

Urine volume and excretion of cyclic AMP, cyclic GMP, prostaglandin E2 (PGE2), thromboxane B2 (TxB2) and creatinine were evaluated as potential indicators of radiation damage in mice given 2-5 Gy to the whole body from an enhanced neutron field. In general, urinary cyclic AMP, cyclic GMP, creatinine and urine volumes were positively correlated across time postexposure, for each radiation dose. TxB2 levels positively correlated with urine volume and cyclic AMP excretion only in animals given 2.0 Gy. None of these parameters suggests their use as a prognostic indicator of the extent of radiation damage. Urinary excretion of PGE2 was negatively correlated with other urinary parameters. Biphasic increases in urinary PGE2 were also observed. The initial transient elevation 2-3 days postexposure was not correlated with the dose (2-5 Gy). The second elevation of PGE2 excretion occurred at 6-10 days. The magnitude of the latter increase suggests that urinary PGE2 excretion may be a useful indicator of whole-body or kidney exposure to neutron fields.     

Effects of ozone on lung mechanics and cyclooxygenase metabolites in dogs.

To determine if acute exposure to ozone can cause changes in the production of cyclooxygenase metabolites of arachidonic acid (AA) in the lung which are associated with changes in lung mechanics, we exposed mongrel dogs to 0.5 ppm ozone for two hours. We measured pulmonary resistance (RL) and dynamic compliance (Cdyn) and obtained methacholine dose response curves and bronchoalveolar lavagate (BAL) before and after the exposures. We calculated the provocative dose of methacholine necessary to increase RL 50% (PD50) and analyzed the BAL for four cyclooxygenase metabolites of AA: a stable hydrolysis product of prostacyclin, 6-keto-prostaglandin F1 alpha (6-keto-PgF1 alpha); prostaglandin E2 (PgE2); a stable hydrolysis product of thromboxane A2, thromboxane B2 (TxB2); and prostaglandin F2 alpha (PgF2 alpha). Following ozone exposure, RL increased from 4.75 +/- 1.06 to 6.08 +/- 1.3 cm H2O/L/sec (SEM) (p less than 0.05), Cdyn decreased from 0.0348 +/- 0.0109 TO .0217 +/- .0101 L/cm H2O (p less than 0.05), and PD50 decreased from 4.32 +/- 2.41 to 0.81 +/- 0.49 mg/cc (p less than 0.05). The baseline metabolite levels were as follows: 6-keto PgF1 alpha: 96.1 +/- 28.8 pg/ml; PgE2: 395.8 +/- 67.1 pg/ml; TxB2: 48.5 +/- 11.1 pg/ml; PgF2 alpha: 101.5 +/- 22.6 pg/ml. Ozone had no effect on any of these prostanoids. These studies quantify the magnitude of cyclooxygenase products of AA metabolism in BAL from dog lungs and demonstrate that changes in their levels are not prerequisites for ozone-induced changes in lung mechanics or airway reactivity.     

Lanthanum stimulates the accumulation of cyclic AMP and inhibits secretion and thromboxane B2 formation in human platelets

La3+ was found to inhibit the secretion of 5-hydroxytryptamine and the production of thromboxane B2 by washed platelets exposed to collagen or thrombin. In addition, La3+ inhibited secretion in response to sodium arachidonate, although the conversion of arachidonate to thromboxane B2 was not affected. La3+ was also found to enhance the accumulation of cyclic AMP under basal conditions and in response to prostaglandin E1, in washed platelets. The inhibition of cyclic AMP accumulation by ADP was prevented by La3+, suggesting that the effect of ADP on cyclic AMP metabolism was dependent upon the presence or flux of calcium at the platelet membrane. La3+ inhibited the activity of adenylate cyclase in platelet lysates both in response to prostaglandin E1 and to F-, indicating a possible effect at the catalytic subunit of the enzyme. None of the observed effects of La3+ could be reversed by the addition of Ca2+ up to 10 mM. The stimulation of cyclic AMP production by La3+ may largely explain the inhibitory effect of La3+ upon platelet secretion and thromboxane B2 production. These results also suggest that Ca2+ localised at the platelet plasma membrane may be important in the regulation of cyclic AMP metabolism.     

Differential induction of apoptosis in x-irradiated L5178Y sublines bearing p53 mutation

We examined apoptosis and expression of p53, E2F-1, bax, bclx_L and bcl2 proteins in two L5178Y (LY) murine lymphoma sublines, LY-R and LY-S, which differ in radiosensitivity and double-strand break (DSB) repair. Both sublines are heterozygous for a p53 mutation in codon 170 that precludes the transactivation function. Accordingly, there is no G1/S arrest after irradiation.We found that there is no change in expression of E2F-1, bax, bclx_L or bcl2 proteins in both LY sublines after x-irradiation. LY-R cells do not constitutively express bcl2, whereas both sublines show high bax content. Radiation induces delayed apoptosis to a greater extent in LY-S than in LY-R cells. The apoptosis can be seen 24 h after irradiation (2 Gy) of LY-S cells, with a maximum at 48 h. LY-R cells need 5 Gy and 72 h post-irradiation incubation to show marked apoptosis (identified by the TUNEL method). The reported observations support the assumption that differential radiosensitivity of LY sublines is associated with the induction of apoptosis that is not related to transactivation by p53 and is primarily related to differential DNA repair ability. Received: 19 August 1999 / Accepted in revised form: 30 November 1999     

Protection of glomerular filtration rate by the thromboxane receptor antagonist L655,240 during low dose cyclosporine administration

Cyclosporin A (CsA) alters the production of prostaglandins (PG) by the kidney. CsA causes an increase in renal vascular resistance, a decrease in renal blood flow, a decrease in glomerular filtration rate (GFR), and increases the renal production of the vasoconstrictor thromboxane. Recently, low dose CsA has been utilized in the treatment of refractory autoimmune diseases. To determine if low dose CsA administration could produce renal hemodynamic alterations and to determine if the thromboxane receptor antagonist L655,240 could prevent these alterations, we administered groups of rats either CsA, 5 mg/kg, subcutaneously and the L655,240 vehicle NaHCO₃ (CsA-NaHCO₃), or CsA and L655,240 (CsA-L655,240), or CsA vehicle and L655,240. The rats were administered the drugs for 7 days and then subjected to inulin and PAH clearances or kidneys were harvested for prostaglandin production studies. CsA significantly depressed GFR and renal plasma flow when compared to the L655,240 treated groups. There was no difference in inulin or PAH clearance between the CsA-L655,240 and CsA vehicle L655,240 groups. Glomerular prostaglandin production including thromboxane was depressed by CsA administration. No histologic alterations were noted in the glomeruli or the medullary portions of the kidney. We conclude that administration of low dose CsA, 5 mg/kg, for 7 days results in a decrease in renal blood flow and GFR without histologic alterations. Administration of the thromboxane receptor antagonist L655,240 prevents the renal hemodynamic alterations induced by CsA in this rat model.     

Prostaglandin E2, prostaglandin E1 and thromboxane B2 release from human monocytes treated with C3b or bacterial lipopolysaccharide

C3b or lipopolysaccharide treatment of human peripheral blood monocytes in culture stimulates an early release of thromboxane B₂ and a delayed release of prostaglandin E into culture supernatants. Immunoreactive thromboxane B₂ release is maximal from 2–8 h, whereas prostaglandin E release is maximal from 16–24 h after stimulation of monocytes in culture. We further examined this process by comparing the time course of labelled prostaglandin E₂, prostaglandin E₁ and thromboxane B₂ release from human monocytes which were pulse or continuously labelled with [³H]arachidonic acid and [¹⁴C]eicosatrienoic acid. The release of labelled eicosanoids was compared with the release of immunoreactive prostaglandin E and thromboxane B₂. The time course of prostaglandin E₂ release was virtually identical to the release of prostaglandin E₁ in all culture supernatants regardless of labelling conditions. However, release of immunoreactive prostaglandin E paralleled the release of labelled prostaglandin E₁ and E₂ only for continuously labelled cultures. The release of labelled prostaglandin E₁ and E₂ from pulse labelled cultures paralleled the release of thromboxane B₂ and not immunoreactive prostaglandin. In contrast, labelled and immunoreactive thromboxane B₂, quantitated in the same culture supernatants, demonstrated similar release patterns regardless of labelling conditions. These findings indicate that the differential pattern of prostaglandin E and thromboxane B₂ release from human monocytes is not related to a time-dependent shift in the release of prostaglandin E₁ relative to prostaglandin E₂. Because thromboxane B₂ and prostaglandin E₂ are produced through cyclooxygenase mediated conversion of arachidonic acid, these results further suggest that prostaglandin E₂ and thromboxane B₂ are independently metabolized in human monocyte populations.     

Protection of glomerular filtration rate by the thromboxane receptor antagonist L655,240 during low dose cyclosporine administration.

Cyclosporin A (CsA) alters the production of prostaglandins (PG) by the kidney. CsA causes an increase in renal vascular resistance, a decrease in renal blood flow, a decrease in glomerular filtration rate (GFR), and increases the renal production of the vasoconstrictor thromboxane. Recently, low dose CsA has been utilized in the treatment of refractory autoimmune diseases. To determine if low dose CsA administration could produce renal hemodynamic alterations and to determine if the thromboxane receptor antagonist L655,240 could prevent these alterations, we administered groups of rats either CsA, 5 mg/kg, subcutaneously and the L655,240 vehicle NaHCO3 (CsA-NaHCO3), or CsA and L655,240 (CsA-L655,240), or CsA vehicle and L655,240. The rats were administered the drugs for 7 days and then subjected to inulin and PAH clearances or kidneys were harvested for prostaglandin production studies. CsA significantly depressed GFR and renal plasma flow when compared to the L655,240 treated groups. There was no difference in inulin or PAH clearance between the CsA-L655,240 and CsA vehicle L655,240 groups. Glomerular prostaglandin production including thromboxane was depressed by CsA administration. No histologic alterations were noted in the glomeruli or the medullary portions of the kidney. We conclude that administration of low dose CsA, 5 mg/kg, for 7 days results in a decrease in renal blood flow and GFR without histologic alterations. Administration of the thromboxane receptor antagonist L655,240 prevents the renal hemodynamic alterations induced by CsA in this rat model.     

Termination of second trimester pregnancy with intra-amniotic 15 (S) 15 methyl prostaglandin F-2alpha - a two dose schedule study.

Termination of second trimester pregnancy with intra-amniotic administration of 15 (S) 15 methyl prostaglandin F-alpha (15 me F-alpha) was attempted in fifty patients. One group (26 patients) was given 1 mg of the analogue and the other group received 2.5 mg. The abortifacient efficacy of 15 me F-2alpha was similar in both groups; over 90% of the patients aborted with a single dose. There was a higher incidence of vomiting, diarrhoea and incomplete abortions in the group treated with 2.5 mg 15 me F-2alpha. Although the mean injection-abortion interval in the 2.5 mg group was shorter, it is concluded that intra-amniotic administration of 1 mg 15 me F-2alpha provides a better regime, giving high efficacy with a single dose, a low incidence of side effects and greater safety in case of inadvertent entry of the intra-amniotic dose into systemic circulation.     

The effect of acute hypoxia on prostaglandin release in perfused human fetal placenta

The release of prostaglandin E2 and F2 alpha, thromboxane B2 and 6-keto-prostaglandin F1 alpha was measured in isolated human placental cotyledons perfused under high- and low-oxygen conditions. Also the effect of reoxygenation on prostaglandin production was studied. During the high-oxygen period, prostaglandin E2 accounted for 44% and 6-keto-prostaglandin F1 alpha for 28% of all prostaglandin release, and the rank order of prostaglandin release was E2 greater than 6-keto-prostaglandin F1 alpha greater than thromboxane B2 greater than prostaglandin F2 alpha. Hypoxia had no significant effect on quantitative prostaglandin release, but the ratio of prostaglandin E2 to prostaglandin F2 alpha was significantly increased. After the hypoxic period during reoxygenation the release of 6-keto-prostaglandin F1 alpha was significantly decreased, as was the ratio of 6-keto-prostaglandin F1 alpha to thromboxane B2. Also the ratio of the vasodilating prostaglandins (E2, 6-keto-prostaglandin F1 alpha) to the vasoconstricting prostaglandins (thromboxane B2, prostaglandin F2 alpha) was decreased during reoxygenation period. With the constant flow rate, the perfusion pressure increased during hypoxia in six and was unchanged in three preparations. The results indicate that changes in the tissue oxygenation in the placenta affect prostaglandin release in the fetal placental circulation. This may also have circulatory consequences.     

Products, kinetics, and substrate specificity of homogeneous thromboxane synthase from human platelets: development of a novel enzyme assay

Homogeneous thromboxane synthase from human platelets converted prostaglandin H2 (PGH2) to thromboxane A2 (measured as thromboxane B2, TxB2), 12(L)-hydroxy-5,8,10-heptadecatrienoic acid (HHT), and malondialdehyde (MDA) in equimolar amounts under a variety of experimental conditions. PGG2 was transformed to MDA and corresponding 15- and 12-hydroperoxy products. PGH1 was enzymatically transformed into 12(L)-hydroxy-8,10-heptadecadienoic acid (HHD) and PGH3 into TxB3 and 12(L)-hydroxy-5,8,10,14-heptadecatetraenoic acid (delta 14-HHT) as earlier reported for solubilized and partially purified thromboxane synthase preparations. The ratio of thromboxane to C17 hydroxy fatty acid formation was 1:1 with PGG2, PGH2, and PGH3 as substrates. These results confirm and extend earlier observations with partially purified enzyme that the three products are formed in a common enzymatic pathway (Diczfalusy, U., Falardeau, P., and Hammarstr?m, S. (1977) FEBS Lett. 84, 271-274). A convenient spectrophotometric assay for thromboxane synthase activity measuring the ultraviolet light absorption of the C17 hydroxy acid formed (e.g., HHT) was developed. The validity of the assay was determined employing specific inhibitors for thromboxane synthase. The substrate specificity of thromboxane synthase was determined using this assay. PGG2 and PGH3 showed Vmax and KM values similar to those of PGH2. The KM value of PGH1 was also identical to that of PGH2 but the Vmax value PGH1 was more than twice as high as that of PGH2.     

Regulatory role of cyclic adenosine 3',5'-monophosphate on the platelet cyclooxygenase and platelet function.

Thromboxane A2 plays an important role in arachidonic acid- and prostaglandin H2-induced platelet aggregation. Agents that stimulate platelet adenylate cyclase (prostaglandin I2, prostaglandin I1 and prostaglandin E1) and dibutyryl cyclic AMP inhibit both thromboxane A2 formation and arachidonate-induced aggregation in platelet-rich plasma. Despite complete suppression of aggregation with agents that elevate cyclic AMP, considerable thromboxane A2 is still formed. Prostaglandin H2-induced aggregations which bypass the cyclooxygenase regulatory step are also inhibited by agents that elevate cyclic AMP without any measurable effect on thromboxane A2 production. These data demonstrate that cyclic AMP can inhibit platelet aggregation by a mechanism independent of its ability to suppress the cyclooxygenase enzyme. Parallel experiments with washed platelet preparations suggest that they may be an inadequate model for studying the relationship between the platelet cyclooxygenase and platelet function.     

Effect of prostaglandin I2 and related compounds on vascular permeability response in granuloma tissues

The effects of prostaglandin I2, 6-ketoprostaglandin F1alpha, prostaglandin E1 and thromboxane B2 on the vascular permeability response in rat carrageenin granuloma were studied with the aid of 131I- and 125I-human serum albumin as indicators for the measurement of local vascular permeability. A single injection of 5 microgram of prostaglandin I2 methyl ester or I2 sodium salt into the locus of the granulomatous inflammation elevated local vascular permeability 2.0-2.5 times over the control within 30 min. The potency was equal to that of the positive control prostaglandin E1 which has been known to be the most potent mediator in this index among several candidate prostaglandins for chemical mediator of inflammation. The other prostaglandin and thromboxane B2 tested were essentially inactive.     

Thromboxane increase in irradiated animals is caused by stimulation of cyclooxygenase activity.

The irradiation of whole body of rabbits with a dose of 6.0 Gy causes an increase in thromboxane synthesis from exogenous arachidonic acid. The uptake of [14C]arachidonic acid and the total amount of radioactivity released during collagen stimulated aggregation of platelets are not changed following the exposure of animals. The irradiation changes the relation between released arachidonic acid and synthesized thromboxane. The amount of 12-hydroxyeicosatetraenoic acid remains unchanged. The results indicate that the increase in thromboxane synthesis is not associated with the activation of phospholipase but is caused by stimulation of cyclooxygenase activity.     

Malonaldehyde formation in intact platelets is catalysed by thromboxane synthase.

Imidazole and compound L8027 (selective inhibitors of thromboxane synthase) produced parallel inhibition of malonaldehyde and thromboxane B2 secretion induced by collagen or thrombin in gel-filtered suspensions of human platelets. Comparing the effects of these inhibitors and aspirin on secretion of granule constituents indicated that platelet degranulation depends mainly on thromboxane production; prostaglandin endoperoxides contributed little.     

Thromboxane receptor stimulation inhibits adenylate cyclase and reduces cyclic AMP-mediated inhibition of ADP-evoked responses in fura-2-loaded human platelets.

Stimulation of human platelets with the thromboxane A2 analogue, U46619, after treatment with prostaglandin E1 or forskolin, reduced the inhibition of ADP-evoked Mn2+ influx and the release of Ca2+ from intracellular stores. U46619 decreased the elevated concentration of 3',5'-cyclic AMP in platelets that were pretreated with prostaglandin E1. These results suggest that occupation of prostaglandin H2/thromboxane A2 receptors, like those for other agonists, inhibits adenylate cyclase activity, which can contribute to the promotion of platelet activation.     

WR-2721 inhibition of radiation-induced prostaglandin excretion in rats

Pre-irradiation administration of the radioprotectant drug WR-2721 to rats resulted in a significant reduction in radiation-induced increases in excretion rates of prostaglandins (PGE and PGF2 alpha) and thromboxane (TxB2). In animals not irradiated. WR-2721 did not significantly alter these excretion rates. Dramatic reductions in the levels of urinary PGE and TxB2 were observed following exposure to 9.0 Gy of whole-body, unilateral gamma-radiation in WR-2721-treated animals, whereas changes in PGF2 alpha levels were less pronounced. Radiation-induced diuresis was also significantly depressed in animals given WR-2721 before irradiation. Reduced prostaglandin excretion rates may reflect the general radioprotective capacity of the chemoprotector WR-2721 on the release of prostaglandins from radiation-damaged tissue. The decrease in diuresis may be related to the observed prostaglandin decreases.