The effect of calmodulin inhibitors on synoviocyte phospholipase A2 activity was evaluated. Cells were incubated with [3H]arachidonic acid for 24 hours to label phospholipids. [3H]prostaglandin E2 synthesis was stimulated by Salmonella minnesota lipopolysaccharide (100 μg/ml). Trifluoperazine, 35 μM, reduced lipopolysaccharide-stimulated [3H]prostaglandin E2 synthesis by 50%. In sonicated suspensions of cells, calcium-dependent phospholipase A2 activity was inhibited by trifluoperazine 3–100 pM and by compound 4880 (3 μg/ml). These agents inhibit calmodulin-dependent enzyme activity. The addition of calmodulin, 1 or 2.5 μM, to compound 4880-treated suspensions reversed this inhibition in a dose-dependent manner. Agents which inhibit calmodulin-dependent enzymes can reversibly inhibit synoviocyte phospholipase A2 and thus prostaglandin E2 production.
In this study the effects of the synthetic acetal plasmalogens (AP) C17-AP, C13-AP, C9-AP on isolated rabbit aorta were investigated with emphasis on their putative relationship with endothelium-dependent relaxing factor (EDRF). The various AP were obtained by total synthesis from the related fatty acid chlorides. In isolated rabbit aorta precontracted with phenylephrine (PE, 10(-7) M), C17-AP (greater than 10(-6) M) and C13-AP (greater than 10(-5) M) exerted an endothelium-dependent relaxation (EDR). In contrast, C9-AP was totally devoid of effect. Given 30 or 45 min before PE, C17-AP (greater than 10(-6) M) and C13-AP (greater than 10(-5) M) abolished the carbachol-induced ERD; moreover, following preincubation with C17-AP, enhanced contraction was obtained with carbachol. Similar effects were obtained with preincubation of aortas with both AA(10(-4) M) and A 23 187 (10(-8) M). Conversely preincubation of aortas with carbachol (3X 10(-6) M) and ATP (3 X 10(-5) M) did not significantly modify the carbachol-induced EDR. Atropine (3 X 10(-7) M) did not affect the relaxation induced by C17-AP and C13-AP. The in vivo effects are correlated with the in vitro actions of acetal plasmalogens: C17-AP and C13-AP but not C9-AP, given iv (5 mg/kg) induced a pronounced but transient hypotension which lasted 2-3 min and which was followed by a persistent hypertensive state. Atropine (0.5 and 1 mg/kg; iv) given 5 min before C17-AP inhibited the secondary hypertensive phase. These results show that C17-AP and C13-AP should be added to the list of compounds capable of releasing EDRF.
The in vitro effects of obstetric drugs on 15-hydroxyprostaglandin dehydrogenase activity were investigated. Enzyme activity was inhibited by indomethacin, methylergometrine maleate, Solcoseryl, conjugated estrogen and progesterone, and was activated only by isoxsuprine-HCL. Methylergometrine maleate and isoxsuprine-HCl, which have opposite functions on uterine muscle, also exerted contrary effects on enzyme activity. Inhibitory and stimulatory effects of these drugs suggested that they could directly regulate uterine activity and also indirectly influence the uterus due to their effects on prostaglandin catabolism in vivo.
Growth of BN175, a malignant fibrosarcoma, was correlated with high plasma TXB2 and PGE2 levels. This statistically significant increase was first detected 17 days after inoculation of the tumor, at which time the tumors were 20 mms in diameter. A further increase in tumor size was associated with still higher PGE2 and TXB2 values. At the same time, progressive alterations in platelet function, as measured by ADP-induced platelet aggregation, were observed.6-keto-PGF1α levels remained normal throughout the whole experiment.
Endogenous prostaglandin (PG) concentrations (6oxo PGF1α, PGE, PGF2α) have been measured by radioimmunoassay in the endaeetrium of women with objectively assessed menstrual blood loss (MBL). The concentration of PGE and “total” PG (6oxo PGF1α+PGE+PGF 2a) was greater in the endometrium of those women with heavy menses (median MBL 152ml (range 86,432) n=16) than in those individuals with a normal menstrual loss (MBL 59m1 (18,78) n=18). The concentrations of PGE and PGF2α were similar in each group, but the concentration of 6oxo PGF1α was significantly less than that of both PGE and- PGF 2α. In 19 individuals, specimens of endometrium were incubated or I and 2 hours in modified 199 medium to assess PG release. There was a direct correlation between endogenous PG content and the production of 6oxo PGF1α, PGE, PGF2α and “total” PG in the first tour, which persisted for the second hour with PGF2α and “total” PG. Endometrial PGs may play a role in the mechanism underlying menstruation; however the observed relationship between the prostanoids and MBL will vary with different experimental methods.
Prostaglandin (PG) levels increase dramatically just before parturition in the rat. Coincident with this dramatic increase in uterine PGs is a precipitous decrease in plasma progesterone and enhanced plasma estradiol levels. The purpose of the present study was to mimic the progesterone withdrawal phenomenon in the presence and absence of estradiol in ovariectomized pregnant rats and determine the effects on uterine PGF, PGE, thromboxane B2 (TxB2) and 6-keto-PGF1a (6KF) levels. Rats were ovariectomized on gay 16 of pregnan4 and silastic inserts containing progesterone and estradiol placed s.c. in two groups of rats (I and II) while the third group (III) received progesterone only. On day 19 of pregnancy progesterone was withdrawn from groups II and III and rats sacrificed 0, 6, 12 and 24 hours later. Uterine tissue was assayed for PGs by radioimmunoassay. Progesterone withdrawal in the absence of estradiol (III) administration significantly (p< .05) elevated PGE, TxB2 and 6KF, but not PGF, at the 24 hour period compared to controls (I). When progesterone was withdrawn in the presence of exogenously administered estradiol (II) only PGF showed enhancement (p < .05) over III at the 24 hour period thus indicating a specific effect of estradiol on the PGF metabolic pathway. In conclusion, these data indicate that: (1) progesterone withdrawal is a potent stimulus for uterine PG production and is probably a major contributor to the augmented uterine PGE, TxB2 and 6KF levels at term in the pregnant rat; and (2) progesterone withdrawal in the presence of exogenously administered estradiol enhances uterine PGF production thus indicating a specific effect of estradiol on PGF production.
It has been suggested that the circulating prostacyclin in primarily inactivated by renal NAD+-dependent 15-hydroxyprostaglandin dehydrogenase. Changes in the renal enzyme activity in response to hormones were studied. The renal 15-hydroxyprostaglandin dehydrogenase activity in female rats was lower than that in male rats, and was significantly increased by ovariectomy. The effect of sex steroids on the renal enzyme activity was then studied in ovariectomized rats. Estradiol administration induced a significant decrease in the renal enzyme activity, while testosterone did not show any significant effect. Kinetic parameters for the renal enzyme from control and estradiol-treated groups were compared. An identical apparent Km for prostaglandin E2 was obtained for the enzyme from both groups. Vmax in the treated group was progressively decreased. The enzyme from both groups decayed at the same rate. The results indicated that estradiol might be the major endogenous sex steroid regulating the renal NAD+-dependent 15-hydroxyprostaglandin dehydrogenase activity, and the inhibitory effect of estradiol on the renal enzyme might be due to the inhibition of the enzyme biosynthesis.
The effects of bradykinin, histamine, phosphatidic acid and leukotrienes B4 and C4 on the distribution and release of 14C-arachidonic acid in human keratinocytes in culture were investigated. Bradykinin, histamine, and phosphatidic acid were found to liberate 14C-arachidonic acid from membrane phospholipids, whereas leukotrienes B4 and C4 were ineffective in this respect. The decrease in the labeling of phospholipids was accompanied by increased labeling of the non-phosphorus lipids. The present study suggests that bradykinin, histamine, and phosphatidic acid may interfere with the distribution and release of arachidonic acid in human keratinocytes in culture.
The intra-uterine administration of actinomycin D on Day 10 reduced the output of prostaglandin (PG) F2α (the major PG released) from the Day 15 guinea-pig uterus in vitro by 80 to 85%. PGE2 output was reduced by 50%, while 6-keto-PGF1α output was unaffected. Plasma progesterone levels were high (3 to 15 ng/ml) on Day 15 due to the reduction in uterine PGF2α output. Endometrial PGF2α synthesizing capacity was reduced by 50% by actinomycin D treatment, while endometrial PGE2 and 6-keto-PGF1α synthesizing capacities were unaffected. Oestradiol treatment in vivo did not reverse the inhibitory effects of actinomycin D on uterine PG production.A23187 increased uterine PGF2α, 6-keto-PGF1α and PGE2 outputs irrespective of treatment, indicating that substrate supply was always rate limiting. Actinomycin D inhibited the uterotrophic action of oestradiol indicating that fresh protein synthesis had been inhibited. Overall, this study suggests that increased protein synthesis is involved in stimulating endometrial PGF2α synthesis and release.Previous studies have shown that increases in enzyme activities induced by oestradiol are only secondary events in the stimulation of endometrial PGF2α production. We propose that oestradiol induces the synthesis of a protein (‘lipostimulin’) which, acting on a progesterone-primed uterus, “switches on” endometrial PGF2α synthesis and release by causing the activation of endometrial phospholipase A2.
The effects of leukotrienes on gastric functions have been outlined by recent experimental and clinical studies. Leukotrienes were found to stimulate pepsin secretion, to reduce mucosal blood flow and to interfere with gastric emptying. Lipoxygenase products appear to impair gastric mucosal integrity and to exacerbate the damaging effects of noxious agents such as absolute ethanol. Drugs acting as leukotriene inhibitors may have a place in the future as anti-ulcer agents.
Monoclonal antibodies to thromboxane B2 (TXB2) have been produced and characterized. Both liquid and solid phase radioimmunoassays have been developed using one of these monoclonal antibodies. The two assays gave similar results when used to quantitate TXB2 in 11 serum samples; however the solid phase assay was more sensitive than the liquid phase assay (i.e., 63 pg/ml vs 19 pg/ml) at a B/BO = 90%). Despite a difference in the sensitivity of the two assay systems, the cross-reactivity of the monoclonal antibody for PGD2, PGE2, PGF2 alpha and 6 keto-PGF1 alpha was the same.
The effects of indomethacin on vascular resistance were investigated in the feline mesenteric vascular bed under conditions of constant blood flow. Indomethacin produced mesenteric vasoconstriction that was dose-dependent but was less active than U46619, the thromboxane mimic, angiotensin II, PGF2α and norepinephrine. The vasoconstrictor response to indomethacin was not altered by meclofenamate and phenoxybenzamine. Indomethacin, in a dose that blocked the systemic vasodepressor response to arachidonic acid, did not alter mesenteric vasoconstrictor responses to sympathetic nerve stimulation, norepinephrine, angiotensin II, U46619, and U44609. The present data suggest that in the feline intestinal vascular bed indomethacin has marked vasoconstrictor activity that occurs independent of activation of alpha-adrenoceptors and formation of cyclooxygenase products possessing pressor activity. The present data suggest that cyclooxygenase products do not modulate vasoconstrictor responses as well as the sympathetic nervous system in the intestinal vascular bed of the cat.
The effects of three antioxidants, selenium, vitamin E and vitamin C, on the production of anti-aggregatory prostacyclin (PGI2) by human endothelial cells and lung tissue as well as on the production of pro-aggregatory thromboxane A2 (TxA2) by human platelets and lung tissue in vitro were studied. Selenium had no effect on endothelial PGI2 synthesis but it dose-dependently inhibited the platelet production of TxA2 (84% at 10(-3) mol/l) and productions of PGI2 (64%) and TxA2 (72%) by human lung. Selenium inhibited platelet TxA2 synthesis much less in the presence of exogenous arachidonic acid (AA) (14% instead of 84%), suggesting that selenium exerts its effect predominantly on phospholipase A2. Vitamin E had no effect on endothelial PGI2, pulmonary PGI2 or TxA2 syntheses but it strongly inhibited platelet TxA2 production (50% at 10(-3) mol/l). This inhibition was only partly (from 50% to 30%) counteracted by exogenous AA suggesting that vitamin E affects both phospholipase A2 and a further step(s) in the TxA2 synthetizing cascade. Vitamin C stimulated endothelial cell PGI2 production (80% at 10(-2) mol/l) but it could not counteract the inhibition of PGI2 production (83%) exerted by 10(-2) mol/l tert-butyl hydroperoxide (TBHP), a promoter of lipid peroxidation. Vitamin C had no effect on pulmonary PGI2 production but it inhibited pulmonary TxA2 formation (38% at 10(-3) mol/l). Slight inhibition of platelet TxA2 production from endogenous AA (6%) turned to slight stimulation (8%) with exogenous AA. The in vivo significance of these results is not yet known.
There have been conflicting reports on the effect of non-steroidal anti-inflammatory drugs on the abortifacient effect of prostaglandins. The efficacy of intra-amniotic 1.5 mg 15(S) 15 methyl PGF2 alpha (15 me PGF2 alpha) in terminating mid-trimester pregnancy in 10 subjects has been compared with the effect of the same medication given to 10 others who had had indomethacin 25 mg and 50 mg orally three and one hour before the PG administration. In the subjects who had the PG analogue only the injection-abortion interval was 17.0 hours. This was significantly shorter (P less than 0.01) than in those who had also received indomethacin (25.4 hours).
Vascular endothelium is a target for leukotriene C4 (LTC4) as demonstrated by previous in vivo and culture experiments. Binding assays were carried out at 0°C on membrane fraction obtained from bovineaortic endothelial cells in culture. Specific binding sites (Kd = 49.9 ± 6.3 nmol × I−1, N = 1.2 × 106 sites per cell) for LTC4 were demonstrated in this preparation. Competition studies showed that LTB4, LTD4 and LTE4 did not displace LTC4 from its binding sites. FPL 55712, a sulfidopeptide antagonist, was seen to be a weak competitor and reduced glutathione exhibited a significant affinity for the binding site. The possible receptor role of this she is discussed.
To determine the effect of anti-neoplastic chemotherapy on the vascular system(s) of children with leukemia/lymphoma, urinary excretion of 6-keto-PGF1α was measured by radioimmunoassay (RIA).
The effect of phenobarbital administration on serum lipoproteins and thromboxane A2 synthesis in platelets was studied in rats. Phenobarbital decreased the serum LDL level by 33% and increased the HDL level by more than 15%. The synthesis of thromboxane A2 in the platelets of the phenobarbital treated animals was found to be reduced by 43%. Thromboxane A2 synthesis in the platelets of the control animals was inhibited by HDL and stimulated by LDL. Hence it appears that the decreased thromboxane A2 synthesis in the platelets of phenobarbital treated rats was at least partly due to the increased HDL and decreased LDL in the serum. Phenobarbital treatment also caused a 15% increase in the serum HDL-cholesterol although it did not have any significant effect on the total serum cholesterol.
Oral administration of two nonsteroidal anti-inflammatory drugs, ibuprofen and flurbiprofen, can suppress the Salmonella-induced arthritis in rats. The joint swelling index of arthritic paws showed suppression of arthritis in animals treated with the drugs, this effect being greater with flurbiprofen. Measurement of 5 eicosanoids in femoral vein plasma showed increase of arachidonic acid products in Salmonella-treated rats. Inhibition of joint inflammation resulting from treatment with ibuprofen and flurbiprofen is reflected by a decrease in concentration of all 5 eicosanoids which were found in the order: PGE2 > TXB2 > 6-keto-PGF1α > PGE1 > PGF2α. These studies indicate that flurbiprofen is a more powerful anti-inflammatory agent than ibuprofen. However, since the joint disease was not completely cured, optimal intervention is quite likely to require modulation of the lipoxygenase pathway.
The existence of diurnal variation in renal function is well described. Prostaglandins are intimately involved with renal physiology, yet a diurnal variation in their excretion is not well documented. We collected 12 consecutive 2 hour urine specimens from 10 young healthy females and measured prostaglandin E2 [PGE2], thromboxane B2 [TXB2], and 6-keto-prostaglandin-F1-alpha by radioimmunoassay for each specimen. We also measured urine volume, urine sodium, and urine creatinine levels. Regression analysis was used to determine the best sine curve for time versus each set of mean values. Only the urinary excretion of PGE2 and TXB2, as well as water were found to significantly fit the generated sine curves. The curves for PGE2 and TXB2 showed a temporal dissociation in their peak and trough values. The excretion of PGE2 between 0800 hours and 2000 hours was significantly higher than during the hours of 2000 and 0800. The opposite was true for the TXB2 excretion. This data suggests that these two prostaglandins and water are excreted in a sine wave pattern. It also suggests that the excretion of PGE2 and TXB2 may respond to different time associated stimuli. We also showed a significant correlation between PGE2 excretion and both the excretion of water and sodium.
