Influence of chemotherapeutic agents on prostacyclin synthesis. II. Effects of tetracycline and penicillin G on prostacyclin synthesis by the rat thoracic aorta and myometrial tissues

The influence of the two antibiotics tetracycline hydrochloride (T) and penicillin G sodium (P) on PGI2 synthesis by the male rat thoracic aorta and day-20 pregnant rat myometrium was investigated in vitro using a rat platelet antiaggregatory bioassay method. Pretreatment of the tissues for 30 min at 37 degrees C with T (21-168 microM) or P (28-224 microM) significantly inhibited PGI2 synthesis in absence or presence of exogenous arachidonic acid (AA) (16.6 microM), (P less than 0.01, n = 5-6). Furthermore, pretreatment of rats with the two drugs (T 11 and P 175 mu mole kg-1 for 30 min) significantly antagonised AA (4 n mole kg-1)-induced hypotension in urethane-anaesthetised rats. They also (T 0.5-4 and P 1-6 microM) antagonised AA-induced aggregation in rabbit citrated platelet-rich plasma. T failed to affect ADP-induced aggregation to any significant level whereas P (3-6 microM) reduced ADP-induced aggregation. The drugs seemed to interfere with the action of the PG endoperoxide synthase (or PG cyclooxygenase) enzyme resulting in decreased formation of PGG2 and PGH2. Such an effect may have resulted from the induced formation of toxic [OH-] radicals and/or inhibition of O2 uptake by the tissues under the influence of the drugs. The demonstrated inherent property of these two antibiotics to inhibit the synthesis of the potent vasodilator, platelet antiaggregatory, anticonvulsant and inhibitor of gastric acid secretion--PGI2, may partly contribute towards better understanding of the biochemical mechanisms that underlie some of the previously known but poorly understood actions of these antibiotics. Furthermore, since good evidence exists for the involvement of excessive uterine prostaglandin synthesis in dysmenorrhoea and premature deliveries, it is suggested that the potential benefits of T or P in these two disorders be investigated.     

Biomarkers of intake for coffee,tea, and sweetened beverages

Non-alcoholic beverages are important sources of nutrients and bioactive compounds that may influence human health and increase or decrease the risk of chronic diseases. A wide variety of beverage constituents are absorbed in the gut, found in the systemic circulation and excreted in urine. They may be used as compliance markers in intervention studies or as biomarkers of intake to improve measurements of beverage consumption in cohort studies and reveal new associations with disease outcomes that may have been overlooked when using dietary questionnaires. Here, biomarkers of intake of some major non-alcoholic beverages—coffee, tea, sugar-sweetened beverages, and low-calorie-sweetened beverages—are reviewed. Results from dietary intervention studies and observational studies are reviewed and analyzed, and respective strengths and weaknesses of the various identified biomarkers discussed. A variety of compounds derived from phenolic acids, alkaloids, and terpenes were shown to be associated with coffee intake and trigonelline and cyclo(isoleucylprolyl) showed a particularly high specificity for coffee intake. Epigallocatechin and 4′-O-methylepigallocatechin appear to be the most sensitive and specific biomarkers for green or black tea, while 4-O-methylgallic acid may be used to assess black tea consumption. Intake of sugar-sweetened beverages has been assessed through the measurement of carbon-13 enrichment of whole blood or of blood alanine in North America where sugar from sugarcane or corn is used as a main ingredient. The most useful biomarkers for low-calorie-sweetened beverages are the low-calorie sweeteners themselves. Further studies are needed to validate these biomarkers in larger and independent populations and to further evaluate their specificity, reproducibility over time, and fields of application.     

Assay of prostacyclin synthesis in intact aorta by aqueous sampling

Conversion of 1-¹⁴C-arachidonic acid (AA) to 6-keto-PGF_1α, the stable metabolite of prostacyclin (PGI₂) was assayed kinetically by employing an aqueous sampling technique. In this way, one can arrive at a kinetic view of PGI₂ synthesis from AA in intact tissue. The assay appears to be particularly suitable to tissues such as the aorta where PGI₂ constitutes the major metabolite of AA. The assay avoids the need for organic solvent extraction and relies on the essential absence of tissue binding of 6-keto-PGF_1α. The disappearance of AA can also be followed in this system but quantitation is complicated by avid tissue binding of the fatty acid. The assay, as described should be applicable to other vascular tissues and should greatly simplify kinetic analyses of prostacyclin synthesis.     

Age-related increase in prostacyclin production in the rat aorta

Normal Sprague-Dawley rats convert a substantial percentage of exogenous arachidonic acid to prostacyclin. This conversion can be quantitated by an aqueous sampling technique utilizing thin layer chromatography and liquid scintillation counting. There is a clear age-related increase in this conversion that can be demonstrated in aortas from rats of 3 weeks to 20 weeks of age.     

Influence of alpha-tocopherol on prostacyclin synthesis by rat's arterial and myometrial tissues

Influence of alpha-tocopherol on PGI2 synthesis by rat arterial and myometrial tissues was investigated using a rat platelet antiaggregatory bioassay. Chronic administration of alpha-tocopherol to female rats (10 mg kg-1 day-1 s.c. for 14 days) significantly increased ex-vivo PGI2 synthesis by the arterial tissue from 12.7 +/- 0.3 (control, mean +/- s.e.m) to 17.2 +/- 0.4 ng PGI2 mg-1 wet tissue and by the myometrial tissue (in proestrus) from 1.1 +/- 0.07 (control) to 1.85 +/- 0.1 ng PGI2 mg-1 wet tissue (P less than 0.05, n = 6). alpha-tocopherol (5 mg kg-1 day-1 for 14 days) did not stimulate PGI2 to any significant level. Pretreatment of male rat arterial tissue with alpha-tocopherol (0.02, 0.1 or 0.2 mM) in vitro increased PGI2 synthesis in a dose-dependent manner. At a dose of 0.2 mM it increased PGI2 synthesis from 13.70 +/- 0.70 (control) to 22.6 +/- 1.4 ng PGI2 mg-1 wet tissue (P less than 0.1, n = 6). Pre-treatment of 14-day pregnant rat myometrium with alpha-tocopherol 0.2 and 0.4 mM significantly increased PGI2 synthesis from 1.2 +/- 0.06 (control) to 1.90 +/- 0.12 and 2.1 +/- 0.1 ng PGI2 mg-1 wet tissue, respectively (P less than 0.05, n = 6). The results indicate that the ability of alpha-tocopherol to stimulate PGI2 synthesis may partly contribute towards better understanding of the mechanisms that underly the protective effect of alpha-tocopherol against experimentally induced decreases in coronary flow and intravascular coagulations in some mammals. Furthermore adequate intake of alpha-tocopherol during pregnancy may enhance uterine blood flow and ensure adequate foetal nutrition.     

Consequences of long-term selenium-deficient diet on the prostacyclin and thromboxane release from rat aorta

It is known that peroxides, which are increased during Se deficiency because of reduced glutathione peroxidase (GSH-Px) activity, can influence the prostacyclin I₂/thromboxane A₂ (PGI₂/TXA₂) ratio. In this study we analyzed the PGI₂ and TXA₂ formation of aortas of long-term Se-deficient rats. Despite low GSH-Px activity in the Se-deficient group, the basal PGI₂ and TXA₂ formation was not different versus control animals (PGI₂: 2295 ± 1134 pg/mg vs 2940 ± 1134 pg/mg; TXA₂: 3.83 ± 1.06 pg/mg vs 5.67 ± 2.99 pg/mg). However, we checked the capacity of the aortas of Se-deficient rats to compensate for a suddenly increased peroxide concentration. After peroxide stimulation, the PGI₂ release was significantly lower in the Se-deficient group compared to the control group (PGI₂: 3507 ± 1829 pg/mg vs 7986 ± 2636 pg/mg). Again, the TXA₂ release did not show any differences. The release ratio of PGI₂/TXA₂ decreased under peroxide stress in Se-deficient animals. Although long-term Se deficiency showed a relatively well-balanced metabolism under resting conditions, sudden stress, accompanied by an excessive radical production, cannot be compensated.     

High density lipoprotein can modulate the inhibitory effect of oxLDL on prostacyclin generation by rat aorta in vitro

To examine the effect of oxidized low density lipoprotein (oxLDL) on prostacyclin (PGI2) generation by rat aorta in vitro and whether high density lipoprotein (HDL) has any protective effect against the inhibition of PGI2 generation induced by oxLDL is the objective of this study. Preincubation of aortas with oxLDL resulted in significant inhibition of PGI2 generation compared to preincubation with normal low density lipoprotein (nLDL) or buffer only. The inhibitory effect of oxLDL resided in its lipid moiety while the lipid fraction of nLDL showed no effect. Aortas preincubated with 10 microg/ml of lyso phosphatidycholine (lyso PC) also showed 30% inhibition of PGI2 generation, indicating that lyso PC was among the lipid components of oxLDL which inhibited PGI2 generation. Preincubation of aortas with a mixture of HDL and oxLDL at a ratio of 10:1 showed a significant recovery of PGI2 generation compared to aortas preincubated with only oxLDL, indicating a protective role for HDL. When HDL was incubated with oxLDL the transfer of lyso PC from oxLDL to HDL suggested that HDL trapped lyso PC from oxLDL thus preventing it from acting on the aorta. However, when a mixture of HDL and oxLDL at a ratio of 3:1 was preincubated with aortas, no protective effect of HDL was observed. Preincubation of aortas with a mixture of HDL plus oxLDL at a ratio of 8:1, which was incubated for 1 h at 37 degrees C, produced significantly less PGI2 than aortas preincubated only with oxLDL, indicating that HDL under these conditions was not protective but even enhanced the inhibitory effect of oxLDL. Similarly, aortas preincubated with HDL plus whole oxLDL (at a ratio of 10:1); containing all the small molecular weight oxidation products and characterized by high levels of thiobarbituric acid reactive substance (TBARS) and lipid hydroperoxides; produced significantly less PGI2 than aortas preincubated with whole oxLDL. These results were evaluated in light of possible modification of HDL by oxLDL and its lipid oxidation products such as aldehydes and lipid peroxides. The modified HDL can add more lipid peroxides and increase the effectiveness of lipid peroxides originally present in oxLDL.     

Acetylcholine induces vasodilation and prostacyclin synthesis in rat lungs

Acetylcholine causes pulmonary vasodilation, but its mechanism of action is unclear. We hypothesized that acetylcholine-induced pulmonary vasodilation might be associated with prostacyclin formation. Therefore, we used isolated rat lungs perfused with a recirculating cell- and plasma-free physiological salt solution to study the effect of acetylcholine infusion on pulmonary perfusion pressure, vascular responsiveness and lung prostacyclin production. Acetylcholine (20 ug infused over 1 minute) caused immediate vasodilation during ongoing hypoxic vasoconstriction and prolonged depression of subsequent hypoxic and angiotensin II-induced vasoconstrictions. Both effects of acetylcholine were abolished by atropine pretreatment. The prolonged acetylcholine effect, but not the immediate response, was blocked by meclofenamate, an inhibitor of cyclooxygenase. The prolonged effect, but not the immediate response, of acetylcholine was associated with an increase in perfusate 6-keto-PGF_1α concentration. The acetylcholine stimulated increase in 6-keto-PGF_1α production was inhibited by meclofenamate and by atropine. Thus, blockade of prostacyclin production corresponded with blockade of the prolonged acetylcholine effect. In conclusion, acetylcholine caused in isolated rat lungs an immediate vasodilation and a prolonged, time-dependent depression of vascular responsiveness. Whereas both acetylcholine effects were under muscarinic receptor control, only the prolonged effect depended on the cyclooxygenase pathway and, presumably, protacyclin synthesis.     

Acetylcholine induces vasodilation and prostacyclin synthesis in rat lungs

Acetylcholine causes pulmonary vasodilation, but its mechanism of action is unclear. We hypothesized that acetylcholine-induced pulmonary vasodilation might be associated with prostacyclin formation. Therefore, we used isolated rat lungs perfused with a recirculating cell- and plasma-free physiological salt solution to study the effect of acetylcholine infusion on pulmonary perfusion pressure, vascular responsiveness and lung prostacyclin production. Acetylcholine (20 micrograms infused over 1 minute) caused immediate vasodilation during ongoing hypoxic vasoconstriction and prolonged depression of subsequent hypoxic and angiotensin II-induced vasoconstrictions. Both effects of acetylcholine were abolished by atropine pretreatment. The prolonged acetylcholine effect, but not the immediate response, was blocked by meclofenamate, an inhibitor of cyclooxygenase. The prolonged effect, but not the immediate response, of acetylcholine was associated with an increase in perfusate 6-keto-PGF1 alpha concentration. The acetylcholine stimulated increase in 6-keto-PGF1 alpha production was inhibited by meclofenamate and by atropine. Thus, blockade of prostacyclin production corresponded with blockade of the prolonged acetylcholine effect. In conclusion, acetylcholine caused in isolated rat lungs an immediate vasodilation and a prolonged, time-dependent depression of vascular responsiveness. Whereas both acetylcholine effects were under muscarinic receptor control, only the prolonged effect depended on the cyclooxygenase pathway and, presumably, prostacyclin synthesis.     

Mutagens in coffee and tea.

Coffee prepared in the usual way for drinking contains a substance(s) that is mutagenic to Salmonella typhimurium TA100 without mammalian microsomal enzymes. One cup of coffee (200 ml) contains mutagen(s) inducing 1.4-4.6 X 10(5) revertants under standard conditions. Instant coffee too is mutagenic to TA100 and one cup of instant coffee prepared from 1 g of coffee powder and 200 ml of water induced 5.6-5.8 X 10(4) revertants of TA100. Caffeine-free instant coffee also has similar mutagenicity. Addition of microsomal enzymes abolished the mutagenicity. Black tea, green tea and Japanese roasted tea were also mutagenic to TA100 without S9 mix and one cup of these teas prepared in the ordinary way produced 1.7-3.8 X 10(4) revertants of TA100. Black tea and green tea were also mutagenic to TA98 in the presence of S9 mix after treatment with a glycosidase from Aspergillus niger, hesperidinase. This type of mutagen in one cup of black tea induced 2.4 X 10(5) revertants of TA98.     

Influence of prostacyclin and two metabolites on the contractility of cultured rat heart cells

Heart cells were cultured from newborn rats, and the contractile activity (CA) and beating frequency (BF) were recorded using an electrooptical technique. Myocardial cells were found to be highly sensitive to Prostacyclin (PGI2) since a 10(-11) M concentration increased the BF and CA. Increasing the concentration (2.7 x 10(-10) to 2.7 x 10(-8) M) resulted in a dose-dependent decrease in CA and BF. The stable product of the non-enzymatic degradation of PGI2 (6 Keto PGF1 alpha) was found to be completely ineffective, and the stable product of the enzymatic PGI2 metabolism (6 Keto PGE1) exerted only a dose-dependent (10(-6) to 10(-5) M) positive inotropic effect. PGI2 was also effective in the presence of serum instead of culture medium but the decrease in CA was less marked than in culture medium, probably due to protein-binding of the drug. When the CA was decreased by PGI2, perfusion with the intracellular calcium-releasing and phosphodiesterase inhibiting agent, caffeine, reversed the PGI2-induced negative inotropic effect. These results suggest that PGI2 participates in the regulation of the heart cell contractility. Its metabolite 6 Keto PGEI could also influence heart cell contractility but higher concentrations are needed. Moreover myocardial intracellular calcium availability seems to be influenced by PGI2.     

The bactericidal activity of tea and coffee

Extracts of black tea, green tea, pu-erh tea or coffee inhibited the growth of various bacteria known to cause diarrhoeal diseases. Tea or coffee also showed bactericidal activity against Staphylococcus aureus and Vibrio parahaemolyticus.     

The anti-haemolysin activity of tea and coffee

Extracts of tea and coffee inhibited the haemolytic activities of Staphylococcus aureus α-toxin and Vibrio parahaemolyticus thermostable direct haemolysin (Vp-TDH). Black tea had the strongest anti-haemolysin activity. Green tea was more active than pu-erh tea. Coffee had anti-Vp-TDH activity but not anti-α-toxin activity.     

Conversions of prostaglandin endoperoxides by prostacyclin synthase from pig aorta

Partially purified prostacyclin synthase from pig aorta converted the prostaglandin (PG) endoperoxide PGH2 to prostacyclin (PGI2), and PGH1 to 12-hydroxy-8,10-heptadecadienoic acid (HHD). Both reactions were inhibited by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HP) in a dose-dependent rashion. However, the reactions PGH2 leads to PGI2 and PGH1 leads to HHD appeared to differ: substrate availability was rate limiting in the latter reaction, while the enzyme became rapidly saturated witth PGH2 and a steady rate of prostacyclin formation was observed at higher substrate levels.     

Conversions of prostaglandin endoperoxides by prostacyclin synthase from pig aorta

Partially purified prostacyclin synthase from pig aorta converted the prostaglandin (PG) endoperoxide PGH₂ to prostacyclin (PGI₂), and PGH₁ to 12-hydroxy-8,10-heptadecadienoic acid (HHD). Both reactions were inhibited by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HP) in a dose-dependent fashion. However, the reactions PGH₂ → PGI₂ and PGH₁ → HHD appeared to differ: substrate availability was rate limiting in the latter reaction, while the enzyme became rapidly saturated with PGH₂ and a steady rate of prostacyclin formation was observed at higher substrate levels.     

Copper deficiency depresses rat aortae superoxide dismutase activity and prostacyclin synthesis

Prostaglandin synthesis shows dependence on lipid hydroperoxides and resultant oxygen derived radical formation. In view of the importance of dietary copper in cytosolic copper dependent superoxide dismutase (Cu/Zn SOD) activity and the role of SOD in oxygen radical formation, the influence of dietary copper on prostacylin (PGI2) synthesis and SOD activity in rat aorta was examined. Copper deficient (0.5 micrograms Cu/g diet) rats showed a significant 47% reduction in PGI2 synthesis rates by aortic ring incubations in comparison to copper adequate (6.0 micrograms Cu/g diet) animals. Aortic SOD activity was reduced by 46% in copper deficiency in comparison to copper adequate animals. Marginal dietary copper (1.6 micrograms Cu/g diet) significantly reduced aortic SOD activity by 32% but was without effect on aortic ring incubation PGI2 synthesis. These results indicate that dietary copper deficiency, and the resultant decrease in SOD activity, depresses aortic PGI2 synthesis.     

Arachidonic acid metabolism in isolated rat aorta. Dependence of prostacyclin biosynthesis on extracellular potassium concentration

Slices of rat aorta were incubated in Krebs-Ringer bicarbonate buffer for measurements of immunoreactive 6-ketoprostaglandin F1 alpha, thromboxane (TX) B2, prostaglandin (PG)E2, and PGF2 alpha, and in Tris buffer (pH 9.3) for determination of prostacyclin (PGI2)-like activity. No significant generation of TXB2, PGE2, or PGF2 alpha by rat aortic tissue could be detected. The time-dependent release of 6-keto-PGF1 alpha Krebs-Ringer bicarbonate buffer closely correlated with PGI2 generation in alkaline Tris buffer. During a 30-min incubation period, 6-keto-PGF1 alpha, release was 79.8 +/- 3.3 pmol/mg at a buffer potassium concentration of 3.9 mmol/liter and significantly increased by 23% to 98.3 +/- 8.5 pmol/mg (P less than 0.025) in the absence of potassium in the incubation medium. A smaller decrease in buffer potassium concentration to 2.1 mmol/liter and an increase to 8.8 mmol/liter did not significantly alter aortic 6-keto-PGF1 alpha release. Changes in the incubation buffer sodium concentration from 144 mmol/liter to either 138 or 150 mmol/liter at a constant potassium concentration of 3.9 mmol/liter did not alter the recovery of 6-keto-PGF1 alpha. Our results support the concept that PGI2 is the predominant product of arachidonic acid metabolism in rat aorta. They further show that PGI2 can be recovered quantitatively as 6-keto-PGF1 alpha under the present in vitro conditions. In addition, this in vitro study points to the potassium ion as a modulator of vascular PGI2 synthesis with a stimulation at low potassium concentrations.     

Low prostacyclin synthetase activity of fetal rat aorta. Progressive increase after birth.

Aortae from fetal or 3 weeks old rats produced very small amounts of PGI₂, prostacyclin. This production increased from 4 weeks on, reaching adult values at about ten weeks. This maturation seemed to be predominantly determined by a change in the PGI₂ synthetase system, rather than in arachidonic acid availability, phospholipase or cyclo-oxygenase activity. The anti-oxidant ascorbic acid stimulated prostacyclin production more strongly in adult than in young rat aortae. This finding suggests that the lower production of PGI₂ by young tissues is not due to an enhanced inhibition of prostacyclin synthetase by lipid peroxides.     

Arachidonate incorporation into rat aorta lipid fractions and eicosanoid formation. Effect of prolonged prostacyclin production

Endogenous arachidonic acid (AA) content, incorporation of radiolabelled AA (AA*) into total lipids, main lipid fractions and different phospholipids (PL), and prostanoid formation have been evaluated in fresh (control) rat arteries and in arteries after 180 min of incubation in buffer (exhausted). The results show that PGI2 formation from endogenous AA decreased 90% in exhausted arteries while AA content decreased only 30%. The total AA* incorporation was significantly higher in exhausted arteries than in controls (p less than 0.01). The distribution of AA* in lipids is altered in exhausted arteries; it increases in total PL, particularly in phosphatidylethanolamine, and decreases in phosphatidylcholine and phosphatidylserine + phosphatidylinositol. AA* content was also lower in triglycerides and esterified cholesterol of exhausted arteries than in control arteries. The AA* metabolized to PGI2 was 83% lower in exhausted arteries than in controls, while PGE2 and TXB2 formation were not modified by the exhaustion process. When the effect that longer incubation in plasma (180 min) has on AA metabolism and turnover was evaluated, PGI2 formation from endogenous AA was found to be increased in comparison with arteries incubated for the de same period in buffer, and the changes observed in the distribution of AA in lipid fractions are smaller than those found in buffer-exhausted aortas. The results of the present study indicate that prolonged production of prostanoids leads to an alteration in AA turnover and to an inactivation of the PGI2-forming system. Plasma seems to protect AA metabolism.     

Prostaglandin synthesis by fetal rat bone in vitro: Evidence for a role of prostacyclin

Prostaglandin synthesis by fetal rat bones was examined by thin-layer chromatography of culture media after preincubation with labeled arachidonic acid. Cultures in rabbit complement (non-heat inactivated serum) were compared with cultures in heat-inactivated serum or cultures treated with indomethacin. The major complement-dependent products were PGE₂, PGF_2α and 6-keto-PGF_1α, the metabolite of prostacyclin (PGI₂). Since PGI₂ had not been previously identified in bone its ability to stimulate bone resorption was tested. Repeated addition of PGI₂ stimulated release of previously incorporated ⁴⁵Ca from fetal rat long bones in both short-term and long-term cultures at concentrations of 10⁻⁵ to 10⁻⁹M. Because of the short half life of PGI₂ in solution at neutral pH, we tested a sulfur analog, thiaprostacyclin (S-PGI₂) which was found to be a stimulator of bone resorption at concentrations of 10⁻⁵ to 10⁻⁶M. These studies suggest that endogenous PGI₂ production may play a role in bone metabolism. Since vessels produce PGI₂ it is possible that PGI₂ release may be responsible for the frequent association between vascular invasion and resorption of bone or calcified cartilage in physiologic remodeling and pathologic osteolysis.