Echinococcus granulosus infection of farm dogs of Iran

The non-enzymatic metabolites of prostacyclin (PGI2) and thromboxane A2 (TXA2), 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha) and thromboxane B2 (TXB2), and their 2,3-dinor metabolites, 2,3-dinor-6-keto-PGF1alpha and 2,3-dinor-TXB2, were measured in early morning urine samples in 24 in vitro fertilization (IVF) cycles in 24 women and in 27 women who became pregnant after IVF and embryo transfer (ET). The sum of the non-enzymatic metabolites and their 2,3-dinor metabolites was considered to be a reflection of total PGI2 and total TXA2 production in vivo. Both the ratio of 'total' PGI2/'total' TXA2 and the ratio of the 2,3-dinor metabolites were calculated. TXB2 concentrations showed virtually no change and the ratios of the non-enzymatic metabolites of PGI2 and TXA2 versus their 2,3-dinor metabolites remained relatively constant. As a consequence, the ratio of 2,3-dinor-6-keto-PGF1alpha/2,3-dinor-TXB2 was a close reflection of the ratio of 'total' PGI2/'total' TXA2, although the latter ratio was significantly higher all the time. We conclude that for comparative studies on the balance between PGI2 and TXA2 in IVF cycles and during gestation, the determination of the 2,3-dinor metabolites alone can replace the measurement of all four metabolites.     

Measurements of urinary prostaglandins in young ovulatory women during the menstrual cycle and in postmenopausal women

The purpose of the present work was to study the prostaglandin excretion in young nonpregnant ovulatory women during the menstrual cycle on the one hand and in postmenopausal women on the other hand and to investigate the influence of female sex hormones (estradiol, progesterone) on urinary prostanoid excretion. Urinary excretion rates of prostaglandin E2 (PGE2), 6-keto-PGF1 alpha, thromboxane B2 (TxB2) and their metabolites PGE-M (11 alpha-hydroxy-9, 15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid), 2,3-dinor-6-keto-PGF1 alpha, 2,3-dinor-TxB2 and 11-dehydro-TxB2 were determined by gas chromatography-triple stage quadrupole mass spectrometry (GC/MS/MS) in 41 young non-pregnant women during the follicular phase and during the luteal phase and in 23 postmenopausal women. Excretion rates of all urinary prostanoids were not significantly different in the follicular phase when compared with the luteal phase. In contrast to the young ovulatory women, PGE2 and TxB2 were significantly higher in postmenopausal women. Concerning the other prostaglandins significant differences between these groups of women did not exist. Although serum levels of estradiol and progesterone were different in young and postmenopausal women, sex hormones have not been shown to correlate with prostaglandins. Our data do not suggest sex hormones to be responsible for the difference in the prostaglandin excretion in women of reproductive age and in women in the menopause. Further systematic investigations into age dependency of prostaglandin excretion in women are necessary.     

Establishment of microanalysis of prostaglandin metabolites by GC/MS and its clinical application

We established microdetermination methods of prostaglandin (PG) metabolites by GC-selected ion monitoring (GC-SIM) and applied them to the clinical investigations. At first the microdetermination of delta 17-6-keto-PGF1 alpha, a hydrolyzed metabolite of PGI2, is described. An authentic delta 17-6-keto-PGF1 alpha was prepared from eicosapentaenoic acid (EPA) incubated with a homogenate from the bovine aortic intima. [18O] delta 17-6-Keto-PGF1 alpha was synthesized to obtain an internal standard for GC-SIM of delta 17-6-keto-PGF1 alpha. A good linear response over the range of 10 pg-5 ng was demonstrated. Chromatographic conditions using a MP-65HT column presented nearly baseline separation of delta 17-6-keto-PGF1 alpha and 6-keto-PGF1 alpha. Furthermore, a monoclonal antibody against cis-3-hexen-1-ol was prepared and used to separate and/or concentrate delta 17-6-keto-PGF1 alpha in the human blood sera. Using the prepared immunoaffinity columns of this antibody, delta 17-6-keto-PGF1 alpha was clearly detected in the human blood sera by GC/MS analysis. We were able to detect delta 17-6-keto-PGF1 alpha of the amount ranging from 6 to 26 pg/ml in the human blood plasma. The present method can be applied to the determination of delta 17-6-keto-PGF1 alpha in the human urine and plasma. Diabetes mellitus induces platelet alterations such as hyperaggregation. Variations in PG production seem to be related to this phenomenon but the changes in PG levels remain unclear. So we microanalyzed the 11-dehydrothromboxane B2 (TXB2) and 2,3-dinor-6-keto-PGF1 alpha, which were stable metabolites of TXA2 and PGI2, in the urine and investigated the relationship between the thromboxane/prostacyclin (TX/PGI) ratio and diabetes mellitus. The TX/PGI ratio in the urine of diabetics was higher than that of healthy volunteers. In murine, the TX/PGI ratio of STZ-induced mice was also higher than that of non-induced mice. The ratio of db/db mice also increased with the progress of diabetes mellitus. Furthermore, we investigated the relationship between the retinal vein occlusion (RVO), a thrombotic disease in which the retinal vein is blocked by blood aggregations, and the TX/PGI ratio. The TX/PGI level in patients with the RVO, who were not combine diabetes, was significantly higher than that in healthy volunteers. One of the causes of the RVO may be due to the variation of thromboxane production. This GC-SIM method can be used to determine the TX/PGI ratio in the urine.     

Milk production of Holstein heifers fed either alfalfa or corn silage diets at two rates of daily gain

OBJECTIVES: L-arginine exerts anti-atherosclerotic effects in hypercholesterolaemic rabbits via modulating endogenous NO production. We investigated whether L-arginine inhibits thromboxane formation in vivo and platelet aggregation ex vivo in this animal model. METHODS: The urinary excretion rates of 2,3-dinor-6-keto-PGF1 alpha (major urinary metabolite of PGI2) and 2,3-dinor-TXB2 (major urinary metabolite of thromboxane A2) were used as indicators of platelet-endothelial cell interactions in vivo. Rabbits were fed 1% cholesterol (Cholesterol group, N = 8), 1% cholesterol plus 2.25% L-arginine (Cholesterol + L-arginine, N = 8), or normal rabbit chow (Control, N = 4) for 12 weeks. Urine samples were collected in weekly intervals. At the end of the study period platelet aggregation ex vivo and endothelium-dependent and -independent vascular function of isolated aortic rings in vitro was assessed. RESULTS: Urinary 2,3-dinor-TXB2 excretion significantly increased in the cholesterol group (p < 0.05), and endogenous NO formation (measured as urinary nitrate excretion) decreased (p < 0.05). Both parameters were significantly correlated with each other (R = 0.48, p < 0.01). L-arginine partly restored urinary nitrate excretion and significantly reduced TXA2 production to values even below those in the control group (p < 0.001). Urinary 2,3-dinor-6-keto-PGF1 alpha excretion increased in early hypercholesterolaemia and returned to control values in the second half of the study period. The early increase in urinary 2,3-dinor-6-keto-PGF1 alpha excretion was attenuated by L-arginine. Platelet aggregation was significantly enhanced in cholesterol-fed rabbits and attenuated by dietary L-arginine. L-arginine also improved the impaired endothelium-dependent relaxations to ADP, and normalized the vasoconstrictor effects of 5-HT in isolated aortic rings. CONCLUSIONS: Cholesterol-feeding enhances platelet aggregation and TXA2 formation, and stimulates platelet-endothelial cell interaction in rabbits. These effects are probably due to impaired NO elaboration, as indicated by decreased urinary nitrate excretion. Chronic dietary supplementation with L-arginine elevates systemic NO elaboration and significantly increases the PGI2/TXA2 ratio. It thus beneficially influences the homeostasis between vasodilator and vasoconstrictor prostanoids in vivo.     

Alteration of thromboxane A2/prostacyclin and their effect on spinal cord blood flow in experimental spinal cord injury in rats

In order to document the contribution of Thromboxane (TXA2) and Prostacyclin (PGI2) to the secondary damage following spinal cord injury (SCI) and their effects on spinal cord blood flow (SCBF), the alteration of SCBF, TXB2 and 6-keto-PGF1 alpha concentration in injury site (T13-L1) and adjacent cords (upper: T12, under: L2) were studied using a rat SCI model induced by Allen's weight drop method (50g-cm). The result showed that after SCI the SCBF in injury site significantly reduced during 1-2 hrs and reduced further during 4-8 hrs. The SCBF in adjacent cords also decreased during 4-8 hrs. TXB2 levels significantly increased at 1 hr and reached peak value at 4 hrs. The 6-keto-PGF1 alpha concentration also significantly increased at 1 hr and maintained that level for 24 hrs. The TXB2/6-keto-PGF1 alpha ratio was significantly elevated at 1 hr and reached its peak at 4 hrs after SCI, then gradually decreased to the preinjury level during 8-24 hrs. The negative correlation of SCBF with TXB2 concentration and TXB2/6-keto-PGF1 alpha ratio were appeared. The experimental results indicated that the imbalance of TXB2/6-keto-PGF1 alpha could be the main cause of microcirculatory disturbance and secondary damage in SCI.     

Relation of the levels of systemic prostaglandins and hemodynamic changes present during orthotopic transplant of the liver]

The aim of this study is to investigate the role of prostaglandins (PGI2 and TXA2) in relation with the hemodynamic alterations occuring after graft reperfusion in patients undergoing OLT. A total of 40 patients with liver cirrhosis were studied. Systemic 6-keto-PGF1 alpha and TXB2, stable metabolites of PGI2 and TXA2 respectively, were determined at the radial artery, at four different surgical stages: basal, hepatectomy, anhepatic, and 10 minutes after graft reperfusion. Overall results showed that 6-keto-PGF1 alpha levels were significantly elevated during hepatectomy (1143 +/- 204) when compared to values in the basal stage (p = 0.007). During hepatectomy, 6-keto-PGF1 alpha levels did not correlate to systemic vascular resistance index (SVRI), neither with the cardiac index (IC) nor with the medial arterial pressure (MAP) in the same stage. During the anhepatic stage, only IRVS was inversely correlated with 6-keto-PGF1 alpha levels (p = 0.004): there was no relation with MAP and CI. During reperfusion no correlations were observed between 6-keto-PGF1 alpha levels and MAP, CI or SVRI. We conclude that systemic PGI2 levels are very high in cirrhotic patients undergoing OLT. The absence of correlation between the magnitude of changes in hemodynamic parameters and 6-keto-PGF1 alpha levels during reperfusion of the new liver suggests that other factors must play a role in these hemodynamic changes.     

Effects of very low dose and enteric-coated acetylsalicylic acid on prostacyclin and thromboxane formation and on bleeding time in healthy subjects

OBJECTIVE: Low dose acetylsalicylic acid (ASA) is widely used as an anti-aggregatory agent in the primary and secondary prevention of cardiovascular diseases. In an effort to spare prostacyclin formation and to reduce gastrointestinal side-effects, both very low doses and enteric-coated formulations of ASA have been introduced. However, it still remains unclear whether these different formulations and dosages are equally effective with respect to inhibition of platelet aggregation and thromboxane A2 (TXA2) formation. METHODS: In a randomized study, we therefore investigated the effects of 100 mg ASA plain (p), 100 mg ASA enteric-coated (ec) and 40 mg ASA (p) to 36 healthy male subjects given for 7 days on platelet aggregation and endogenous prostanoid formation rates. Platelet aggregation and platelet TXB2 release in platelet rich plasma (PRP) and serum TXB2 and 6-keto-PGF1alpha levels were determined at baseline and after 7 days of each medication. The urinary metabolites of TXA2 (2,3-dinor-TXB2) and prostacyclin (2,3-dinor-6-keto-PGF1alpha) were measured by gas chromatography/tandem mass spectrometry in 24-h-urines at baseline and on day 7 of each medication. RESULTS: Collagen-induced platelet aggregation was 73.1+/-1.6% of maximal aggregation at baseline. It was inhibited by 68.9%, 58.6% and 24.0% by ASA 100 mg plain, 100 mg enteric-coated, and 40 mg plain on day 7, respectively. Platelet TXB2 release was 11592.0+/-367.5 pg x ml(-1) PRP. It was inhibited by 90.1%, 86.5%, and 55.2% by ASA 100 mg plain, 100 mg enteric-coated, and 40 mg plain, respectively. Serum TXB2 was almost completely reduced on day 7 by 100 mg ASA, but not by 40 mg ASA; serum 6-keto-PGF1alpha was slightly, but significantly reduced in all three groups. Urinary 2,3-dinor-TXB, excretion was 196.0+/-41.5 pg x mg(-1) creatinine at baseline. It was reduced by 80.3% and 79.1% by ASA 100 mg plain and enteric-coated, respectively (each P < 0.05 versus baseline), but only by 55.4% by ASA 40 mg plain (P < 0.05 versus both formulations of ASA 100 mg). CONCLUSIONS: Our present data show that the plain and enteric-coated formulations of 100 mg ASA are equally effective in inhibiting platelet aggregation, platelet thromboxane production, and urinary 2,3-dinor-TXB2 excretion rates. In contrast, a very low dose of 40 mg ASA was significantly less effective in inhibiting these indices of platelet activation in healthy human subjects. ASA enteric-coated 100 mg may be a useful alternative to 100 mg ASA (p) in patients with gastrointestinal side-effects, whereas 40 mg ASA (p) may be too low to inhibit sufficiently platelet activity in patients with cardiovascular diseases in whom platelet activity is increased.     

Effect of captopril on prostacyclin and nitric oxide formation in healthy human subjects: interaction with low dose acetylsalicylic acid

1. Angiotensin converting enzyme inhibitors have been suggested to act in part by potentiating the stimulatory effect of bradykinin on endothelial prostacyclin and/or nitric oxide (NO) formation. This may give rise to interaction with cyclo-oxygenase inhibiting drugs like acetylsalicylic acid, which is most often used in low doses in patients with cardiovascular diseases. 2. We investigated the effects of captopril (2 x 25 mg day-1), or ASA (1 x 100 mg day-1), or the combination of both drugs for 7 days, on blood pressure, prostanoid and NO formation rates in a double-blind, double dummy, randomized crossover study in 13 healthy female subjects. The urinary metabolites of thromboxane A2 (2,3-dinor-TXB2) and prostacyclin (2,3-dinor-6-keto-PGF1 alpha), and PGE2 were measured by gas chromatography/tandem mass spectrometry in urine on days 1, 6 and 7 of each medication. NO formation was assessed using urinary NO3- and cyclic GMP as indicators. 3. Urinary 2,3-dinor-6-keto-PGF1 alpha excretion was not significantly changed by either captopril, ASA, or their combination. Urinary 2,3-dinor-TXB2 excretion was inhibited by > 80% by ASA alone or in combination with captopril (each P < 0.05), but was not affected by captopril alone. Urinary PGE2 excretion was not significantly changed by either of the treatments. Urinary NO3- and cyclic GMP excretion rates were not significantly changed by captopril, ASA, or their combination. 4. Blood pressure was slightly reduced by captopril. ASA had no effect on blood pressure when given alone, nor did it modulate the effect of captopril on blood pressure during co-administration. Angiotensin II/angiotensin I ratio (index of ACE activity) was significantly decreased by captopril alone or in combination with ASA, but was unaffected by ASA alone. 5. Captopril does not stimulate prostacyclin formation in healthy human subjects in a dose sufficient to substantially inhibit ACE activity. Co-administration of ASA significantly inhibits 2,3-dinor-TXB2 excretion, but does not interfere with the blood pressure lowering effect of captopril in healthy human subjects.     

Prostaglandins and thromboxanes in amniotic fluid during rivanol-induced abortion and labour

Abortion or delivery were induced by extra-amniotic instillation of Rivanol during the second trimester in twelve patients and during the third trimester in two patients with fetal death and one patient with fetal acrania. Serial sampling of amniotic fluid was performed through a transabdominal catheter and the levels of free arachidonic acid (AA), prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 (PGE2), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and thromboxane B2 (TXB2) were determined. The levels of AA, PGF2 alpha, PGE2, 6-keto-PGF1 alpha and TXB2 in amniotic fluid increased significantly during induction with the exception of AA in fetal death which was high and remained constant during induction. Furthermore, PGF2 alpha, 6-keto-PGF1 alpha and TXB2 were all significantly correlated to AA. These observations suggested that free AA is released during Rivanol-induction of abortion and labour giving an increased synthesis of PGF2 alpha, PGE2 prostacyclin and thromboxane A2 in the fetal membranes and the decidua but not in the fetus. This increase might be relevant for the initiation and progress of abortion and labour in these patients.     

Alpha adrenergic receptor subtypes involved in prostaglandin synthesis are coupled to Ca++ channels through a pertussis toxin-sensitive guanine nucleotide-binding protein

Previously, we have shown that alpha-2C and alpha-1A adrenergic receptors (AR) stimulate prostacyclin (PGI2) synthesis through a pertussis toxin-sensitive guanine nucleotide-binding protein (G protein) in vascular smooth muscle cells (VSMC). The purpose of this study was to assess the role of Ca++ in PGI2 production elicited by alpha-AR activation and to investigate the modulation of the Ca++ channel by G proteins coupled to these alpha-AR in VSMC. PGI2 was measured as immunoreactive 6-keto-PGF1 alpha by radioimmunoassay and cytosolic calcium ([Ca++]i) by spectrofluorometry using fura-2. Norepinephrine, methoxamine and UK-14304 enhanced 6-keto-PGF1 alpha production and [Ca++]i, which was inhibited by depletion of extracellular Ca++ and by Ca++ channel antagonists (verapamil, nifedipine and PN 200-110). Moreover, the Ca++ channel activator Bay K 8644 increased 6-keto-PGF1 alpha production in a nifedipine-sensitive manner, indicating the involvement of dihydropyridine-sensitive Ca++ channels in VSMC. Pertussis toxin inhibited AR agonist-induced 6-keto-PGF1 alpha production and the increase in [Ca++]i. Alpha AR agonists increase Ca++ influx in the presence of guanosine 5'-0-(2- thiodiphosphate) (GTP-gamma-S), and this effect was blocked in the presence of guanine 5'-O-(2-thiodiphosphate) (GDP-beta-S) and antiserum against Gi alpha 1-2 protein in reversibly permeabilized cells with beta-escin. VSMC of rabbit aortae contain a G protein(s) that was recognized by Gi alpha 1-2 but not Gi alpha 3 or G0 antibodies at 1:200 dilution. The calmodulin inhibitor W-7 blocked AR agonist and Bay K 8644-stimulated 6-keto-PGF1 alpha production. The phospholipase A2 inhibitors 7,7-dimethyleicosadienoic acid and oleoyloxyethyl phosphocholine but not phospholipase C inhibitor U-73122 reduced 6-keto-PGF1 alpha production in VSMC. These data suggest that a pertussis toxin-sensitive G protein, probably Gi alpha 1-2, coupled to alpha AR regulates Ca++ influx, which, in turn, by interacting with calmodulin, increases phospholipase A2 activity to release arachidonic acid for PGI2 synthesis in VSMC of rabbit aortae.     

Beta adrenergic receptor stimulated prostacyclin synthesis in rabbit coronary endothelial cells is mediated by selective activation of phospholipase D: inhibition by adenosine 3'5'-cyclic monophosphate

Activation of beta adrenergic receptors in the isolated rabbit heart by catecholamines stimulates prostacyclin (PGI2) synthesis, which is inhibited by adenosine 3'5'-cyclic monophosphate (cAMP). The purpose of this study was to determine if activation of beta adrenergic receptors in cultured coronary endothelial cells (CEC) of rabbit heart with isoproterenol (ISOP) stimulates PGI2 synthesis and if cAMP inhibits the synthesis of this prostanoid and to investigate the underlying mechanism. Incubation of CEC with ISOP increased production of cAMP and PGI2, measured as immunoreactive cAMP and 6-keto-prostaglandin F1alpha, (6-keto-PGF1alpha), respectively. Forskolin, an activator of adenylyl cyclase, increased cAMP accumulation and inhibited ISOP-stimulated 6-keto-PGF1alpha synthesis. 8-(4-chlorophenyl-thio) cAMP also inhibited ISOP-induced 6-keto-PGF1alpha production. However, miconazole, an inhibitor of adenylyl cyclase, reduced cAMP accumulation and enhanced ISOP-stimulated 6-keto-PGF1alpha synthesis in CEC. ISOP-induced 6-keto-PGF1alpha synthesis was attenuated by C2-ceramide, an inhibitor of phospholipase D (PLD) by propranolol, a beta-AR antagonist that also inhibits phosphatidate phosphohydrolase and by the diacylglycerol lipase inhibitor 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC 80267). Acetylcholine (ACh) induced 6-keto-PGF1alpha synthesis was also inhibited by these agents. Both ISOP and ACh increased PLD activity, which was inhibited by C2-ceramide but not by RHC 80267 or propranolol. ACh but not ISOP increased phospholipase A2 activity in CEC. ISOP- but not ACh-induced increase in PLD activity was attenuated by forskolin and 8-(4-chlorophenyl-thio)-adenosine 3'-5'-cyclic monophosphate and augmented by miconazole. These data suggest that beta adrenergic receptors activation promotes PGI2 synthesis in the CEC by selective activation of PLD and that cAMP decreases PGI2 synthesis by decreasing PLD activity. Moreover, beta adrenergic receptors activated PLD appears to be distinct from that stimulated by ACh.     

Prostacyclin formation elicited by endothelin-1 in rat aorta is mediated via phospholipase D activation and not phospholipase C or A2

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that also stimulates production of prostacyclin (PGI2) from arachidonic acid. The purpose of this study was to determine the contribution of phospholipases (PLs) A2, C, and/or D in ET-1-induced PGI2 formation in the rat aorta, measured as immunoreactive 6-ketoprostaglandin (PG) F1 alpha. ET-1 increased 6-keto-PGF1 alpha formation, which was not affected by a PLA2 inhibitor, 7,7-dimethyl eicosadienoic acid (DEDA). Furthermore, ET-1 failed to stimulate PLA2 activity measured in the cytosol (cPLA2), using phosphatidylcholine, L-a-1-palmitoyl-2-arachidonyl[14C] as a substrate. However, the adrenergic agonist norepinephrine increased 6-keto-PGF1 alpha formation, which was attenuated by DEDA, and enhanced PLA2 activity. ET-1 enhanced PLC activity, as indicated by increased inositol phosphate production, which was prevented by a PLC inhibitor, U-73122. However, ET-1-induced 6-keto-PGF1 alpha production was not altered by U-73122. An inhibitor of PLD activation, C2-ceramide, attenuated ET-1-induced PLD activity, as indicated by the production of phosphatidylethanol. Furthermore, ET-1-induced 6-keto-PGF1 alpha formation was inhibited by C2-ceramide as well as by ethanol treatment. Moreover, inhibitors of phosphatidate phosphohydrolase (propranolol) and diacylglycerol lipase (RHC-80267), attenuated ET-1-induced 6-keto-PGF1 alpha formation. Finally, ET-1-induced activation of PLD was not attenuated by a selective PKC inhibitor, bisindolylmaleimide I. These data suggest a novel pathway for ET-1-induced PGI2 formation in the rat aorta involving activation of PLD but not cPLA2 and independent of PLC or PKC activation.     

The effect of dexamethasone on CRH and prostanoid production from human term placenta

The human placenta at term produces large quantities of corticotropin releasing hormone (CRH) and prostanoids. These hormones play an important role in the maintenance of pregnancy, and the initiation and progress of labor; yet little is known of factors affecting their regulation and the interrelationship of CRH and prostanoid production. In these studies we have investigated the effect of dexamethasone on the production of CRH and prostanoids from fresh human term placental tissues. The basal release of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), thromboxane B2 (TxB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) from human term placental explants increased from the fifth hour in culture, while the release of 13,14-dihydro-15-keto-PGF2 alpha (PGFM) was not significantly changed during this period. The addition of dexamethasone (10(-8) M) to the perifusing medium resulted in a rapid and dramatic inhibition of PGE2, PGF2 alpha, PGFM, TxB2 and 6-keto-PGF1 alpha release. On the other hand, CRH release was not significantly changed throughout the seven hours of incubation with dexamethasone. These data demonstrate that glucocorticoids at physiologic concentrations can inhibit human term placental prostanoid production, and thus glucocorticoid production may play an important role in the physiological regulation of placental prostanoid production in the human placenta. However, dexamethasone did not alter CRH release, demonstrating that the inhibition of placental prostanoids by dexamethasone is not a CRH mediated event.     

Correction of increased prostacyclin synthesis in Bartter's syndrome by indomethacin treatment

The role of prostacyclin and thromboxane A2 in the pathogenesis of Bartter's syndrome was investigated by measurement of the urinary excretion of 6-keto-PGF1 alpha and thromboxane B2, respectively, in five patients. The prostaglandin metabolites were extracted from urine by a reproducible method and measured by specific radioimmunoassays. The patients with Bartter's syndrome excreted about four-times as much 6-keto-PGF1 alpha as the normal controls. In contrast, there was no significant difference in the urinary excretion of thromboxane B2 between the patients and the controls. In a second part of the study, three patients were treated with indomethacin (150 mg/day for four days), an inhibitor of prostaglandin synthesis. This regimen suppressed urinary excretion of 6-keto-PGF1 alpha by 43% and that of thromboxane B2 by 46%. It is suggested that the increase in prostacyclin production is responsible for both the hyperreninemia and and the other endocrine derangements as well as the hyporesponsiveness of blood pressure to intravenous infusion of vasopressors in patients with Bartter's syndrome.     

Increased production of eicosanoids, TXA2, PGI2 and LTC4 in experimental spinal cord injuries

Arachidonate metabolites have many kinds of bioactivities. Thromboxane A2 (TXA2) stimulates platelet aggregation and vasoconstriction, whereas prostaglandin I2 (PGI2) antagonises its activities. Thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) are determined in biological materials. Production of TXB2, 6-keto-PGF1 alpha and leukotriene C4 (LTC4), which have potent vascular permeability, was measured by radioimmunoassay in experimental spinal cord injured animals. TXB2 level in the rat spinal cord reached a peak concentration of 133.6 +/- 3.8 pmol/g cord, and 6-keto-PGF1 alpha increased to 26.2 +/- 11.7 pmol/g cord 5 minutes after the injury. There was good correlation between TXB2 production and vascular damage as monitored by fluorescein uptake. When OKY-046 ((E)-3-[4-(1-imidazolylmethyl) phenyl]-2-propenoic acid), which selectively inhibits TXA2 synthetase activity, was administered 10 minutes before injury, the increase in TXB2 production was inhibited by more than 80%, but the degree of vascular damage was reduced by only 40%. In the guinea pig spinal cord, LTC4 levels reached a peak concentration of 2.2 +/- 0.4 pmol/g cord 10 minutes after compression, while that of TXB2 reached 146.8 +/- 6.2 pmol/g cord. The increased production of TXB2 was correlated with the degree of compression injury while that of LTC4 production did not. These findings suggest that vasoactive eicosanoids, TXA2, PGI2 and LTC4, play important roles in secondary damage following spinal cord injury, although their roles may be different among species of animals.     

Production of prostacyclin and thromboxane in lupus pregnancies: effect of small dose of aspirin

OBJECTIVES: To find out whether the tendency toward poor outcome in lupus pregnancies could be explained by changes in prostacyclin/thromboxane production, to relate these changes to the presence of antiphospholipid antibodies, and to study the potential benefits of low-dose aspirin. METHODS: We followed the urinary output of prostacyclin metabolites (6-keto-prostaglandin [PG]F1 alpha, 2,3-dinor-6-keto-PGF1 alpha) and thromboxane metabolites (thromboxane B2, 2,3-dinor-thromboxane B2) using high-pressure liquid chromatography followed by radioimmunoassay. We studied 14 pregnant women with systemic lupus erythematosus (SLE), of whom six had detectable antiphospholipid antibodies. The patients were randomized by a computerized program to receive either 50 mg aspirin daily (six women) or placebo (eight women). Nine healthy pregnant women served as controls. RESULTS: The production of prostacyclin was normal in early pregnancy in SLE patients but was reduced during late gestation in those without antiphospholipid antibodies. The production of thromboxane was increased in SLE patients compared with controls, and this increase was highest (two-to threefold rise) when antiphospholipid antibodies were detectable. Aspirin eliminated thromboxane dominance without affecting prostacyclin production. CONCLUSION: These data suggest that the presence of antiphospholipid antibodies in SLE patients may trigger thromboxane dominance, possibly contributing to the adverse outcome of these pregnancies. This thromboxane dominance can be eliminated with aspirin.     

Nitric oxide as a regulator of prostacyclin synthesis in cultured rat heart endothelial cells

The effects of nitric oxide (NO) and its second messenger cyclic guanosine monophosphate (cGMT) on prostacyclin (PGI2) synthesis were studied in cultured rat heart endothelial cells using three different non-enzymatic nitric oxide releasing substances as well as inhibitors of nitric oxide synthase and of soluble guanylate cyclase. Production of prostacyclin, measured as 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), was stimulated up to 1.7 fold in endothelial cells treated with the NO donors SIN-1 (3-morpholino sydnonimine), GEA 3162 (3-aryl-substituted oxatriazole imine) and GEA 3175 (3-aryl-substituted oxatriazole sulfonyl), chloride). In each case the synthesis of cGMP increase as much as 40-100 fold. An inhibitor of NO synthase, NG-nitro-L-arginine methyl ester (L-NAME), decreased the basal production of 6-keto-PGF1 alpha in non-stimulated endothelial cells, an effect that could be reversed by the NO donors SIN-1, GEA 3162 and GEA 3175. cGMP formation in the L-NAME treated endothelial cells was unaltered. The guanylate cyclase inhibitors, methylene blue (100 mumol/l) and LY83583 (100 mumol/l), caused a 1.5-10 fold increase in 6-keto-PGF1 alpha production while NO-donor-stimulated endothelial cGMP production was decreased by 10 to 90%. However, when SIN-1 was used as a stimulant, LY83583 had no significant effect on the production of cGMP. These findings support the hypothesis that NO stimulates prostacyclin production directly by activating cyclooxygenase. The results also suggest that NO could have an indirect effect on prostacyclin production via cGMP.     

Cyclooxygenase-1 and -2 of endothelial cells utilize exogenous or endogenous arachidonic acid for transcellular production of thromboxane

The presence of prostaglandin (PG) H2 in the supernatant of human umbilical vein endothelial cells (HUVEC) stimulated by thrombin restores the capacity of aspirin-treated platelets to generate thromboxane (TX) B2. Induction of cyclooxygenase-2 (Cox-2) by interleukin (IL)-1alpha or a phorbol ester increases this formation. HUVEC treated with aspirin lost their capacity to generate PGs but recovery occurred after 3- or 6-h induction of Cox-2 with phorbol ester or IL-1alpha. Enzyme activity of the newly synthesized Cox-2 in aspirin-treated cells, evaluated after immunoprecipitation, was similar to untreated cells but after 18 h of cell stimulation only 50-60% recovery of Cox-1 was observed. The use of SC58125, a selective Cox-2 inhibitor, confirmed these findings in intact cells. Cyclooxygenase activity was related to the amount of Cox proteins present in the cells, but after induction of Cox-2, contribution of the latter to PG production was 6-8-fold that of Cox-1. Aspirin-treated or untreated cells were incubated in the absence or presence of SC58125 and stimulated by thrombin, the ionophore A23187, or exogenous arachidonic acid. The production of endogenous (6-keto-PGF1alpha, PGE2, PGF2alpha) versus transcellular (TXB2) metabolites was independent of the inducer, the source of arachidonic acid and the Cox isozyme. However, in acetylsalicylic acid-treated cells, after 6-h stimulation with IL-1alpha, newly synthesized Cox-2 produced less TXB2 than 6-keto-PGF1alpha compared to untreated cells. At later times (>18 h), there was no metabolic difference between the cells. These studies suggest that in HUVEC, Cox compartmentalization occurring after short-term activation may selectively affect transcellular metabolism, but not constitutive production, of PGs.     

Interleukin-1alpha and tumor necrosis factor alpha synergistically stimulate prostaglandin E2-dependent production of interleukin-11 in rheumatoid synovial fibroblasts

OBJECTIVE: Interleukin-11 (IL-11), an IL-6-type cytokine, is thought to be involved in bone resorption via osteoclast differentiation. Here, we characterized the combined effect of IL-1alpha and tumor necrosis factor alpha (TNFalpha), major cytokines in the rheumatoid synovium, on the production of IL-11 by cultured rheumatoid synovial fibroblasts (RSFs). METHODS: The amounts of IL-11, IL-6, and prostaglandin E2 (PGE2) were measured by enzyme-linked immunosorbent assay. IL-11 messenger RNA (mRNA) levels were determined by Northern blotting. Protein expression of cytosolic phospholipase A2 (cPLA2), cyclooxygenase 2 (COX-2), and protein kinase C (PKC) isoforms were determined by Western blotting. RESULTS: IL-1alpha and TNFalpha synergistically stimulated RSFs to produce IL-11 at both the mRNA and protein levels. This synergistic effect was completely inhibited by indomethacin. The inhibition was prevented by PGE2, indicating that the synergistic effect of IL-1alpha and TNFalpha was PGE2-mediated. The cooperative effects of these 2 cytokines were also observed in the production of PGE2 and the expression of 2 regulatory enzymes in PGE2 production, cPLA2 and COX-2. The synergistic induction of IL-11 by IL-1alpha and TNFalpha was completely inhibited by a potent inhibitor of all isoforms of PKC, GF109203X. In contrast, phorbol myristate acetate, which induced a down-regulation of PKC, degrading all PKC isoforms except atypical PKC, did not affect the induction of IL-11. CONCLUSION: These findings suggest that IL-1alpha and TNFalpha synergistically stimulate the production of IL-11 via their effects on PGE2 production in the rheumatoid joint, and that atypical PKC may be another target for down-regulation of IL-11, the bone resorption-associated cytokine.     

Effects of tangshenkang capsule on diabetic nephropathy

To study the effects of Chinese herbal medicine Tangshenkang (TSK) capsule on diabetic nephropathy (DN), 57 patients with DN were randomly divided into two groups, the treated group and the control group, they were treated with TSK capsule and the conventional therapy respectively. There were serious disorders of metabolism in DN patients, that showed the TXB 2/6-keto-PGF1 alpha ratios and lipid peroxidase (LPO) levels were higher than that of healthy people. After 6 weeks treated with TSK capsule the albuminuria levels reduced obviously (decreased 51%), renal plasma flow (RPF) increased, glomerular filtration rate and the LPO levels decreased and a positive correlation was observed between albuminuria levels and TXB 2/6-keto-PGF1 alpha ratios while the clearance rate of creafinin didn't improve significantly. There were no significant difference in the above-mentioned parameters in the control group before and after treatment. These results suggested that TSK capsule possessed a significant effect in improving albuminuria and glomerular function. And the effect of TSK might be due to its adjusting TXB 2/6-keto-PGF1 alpha ratios and its lipid-peroxidation in DN patients.