Interactions between erythromycin, flunixin meglumine, levamisole and plant secondary metabolites towards bovine gastrointestinal motility-in vitro study.

Continued ingestion of plant secondary metabolites by ruminants can provoke pharmacological interactions with pharmaceutical agents used in animals. As some drugs and phytocompounds affect smooth muscle activity, the aim of this study was to verify the possible interaction between selected pharmaceutical agents and plant secondary metabolites towards bovine gastrointestinal motility. The interactions between phytocompounds-apigenin, quercetin, hederagenin, medicagenic acid-and medicines-erythromycin, flunixin meglumine and levamisole-were evaluated on bovine isolated abomasal and duodenal specimens obtained from routinely slaughtered cows. The obtained results confirmed the contractile effect of all three drugs used solely. Hederagenin and medicagenic acid (0.001 µM) enhanced the contractile effect of levamisole. Hederagenin additionally increased the impact of erythromycin. Both saponins (100 µM) showed synergistic effects with all tested pharmaceuticals. Apigenin and quercetin (0.001 µM) intensified the contractile response induced by erythromycin and levamisole. Moreover, both flavonoids (100 µM) showed an antagonistic interaction with all tested drugs which in that situation were devoid of the prokinetic effect. To conclude, plant metabolic metabolites such as saponins and flavonoids are potent modifiers of the effect of drugs towards gut motility. The synergy observed between phytocompounds and selected medicines can be beneficial in the treatment of cows with hypomotility disorders.

Modification of abomasum contractility by flavonoids present in ruminants diet: in vitro study.

Flavonoid supplementation is likely to be beneficial in improving rumen fermentation and in reducing the incidence of rumen acidosis and bloat. Flavonoids are also said to increase the metabolic performance during the peripartum period. Ruminants are constantly exposed to flavonoids present in feed. However, it is not clear if these phytochemicals can affect the activity of the gut smooth muscle. Therefore, the aim of the study was to verify the effect of three flavonoids on bovine isolated abomasum smooth muscle. The study was carried out on bovine isolated circular and longitudinal abomasal smooth muscle specimens. All experiments were conducted under isometric conditions. The effect of apigenin, luteolin and quercetin (0.001 to 100 µM) was evaluated on acetylcholine-precontracted preparations. The effect of multiple, but not cumulative, treatment and single treatment with each flavonoid on abomasum strips was compared. Apigenin (0.1 to 100 µM) dose-dependently showed myorelaxation effects. Luteolin and quercetin applied in low doses increased the force of the ACh-evoked reaction. However, if used in high doses in experiments testing a wide range of concentrations, their contractile effect either declined (luteolin) or was replaced by an antispasmodic effect (quercetin). Surprisingly, the reaction induced by flavonoids after repeated exposure to the same phytochemical was not reproducible in experiments testing only single exposure of abomasum strips to the same flavonoid used in a high concentration. Taking into account the physicochemical properties of flavonoids, this data suggests the ability of flavonoids to interfere with cell membranes and, subsequently, to modify their responsiveness. Assuming ruminant supplementation with luteolin or quercetin or their presence in daily pasture, a reduction of the likelihood of abomasum dysmotility should be expected.

Participation of cholinergic pathways in α-hederin-induced contraction of rat isolated stomach strips.

The dry extract of Hedra helix leaves and its main active compounds, predominantly α-hederin and hederacoside C, has been traditionally believed to act spasmolytic. However, it has been recently proved that both, the extract of ivy and triterpenoid saponins, exhibit strong contractile effect on rat isolated stomach smooth muscle strips. It turned out that the most potent contractile agent isolated from the extract of ivy leaves is α-hederin. Thus, it seems reasonable to estimate the mechanism of the contractile effect of this saponin. The presented study was aimed at verifying the participation of cholinergic pathways (muscarinic and nicotine receptors) in α-hederin-induced contraction. The experiments were carried out on rat isolated stomach corpus and fundus strips under isotonic conditions. The preparations were preincubated with either atropine or hexamethonium and then exposed to α-hederin. All results are expressed as the percentage of the response to acetylcholine - a reference contractile agent. The obtained results revealed that the pretreatment of isolated stomach strips (corpus and fundus) with atropine neither prevented nor remarkably reduced the reaction of the preparations to α-hederin. Similarly, if the application of saponin was preceded by the administration of hexamethonium, the strength of the contraction of stomach fundus strips induced by α-hederin was not modified. Concluding, it can be assumed that the cholinergic pathways do not participate in α-hederin-evoked contraction of rat isolated stomach preparations.

Enzymatic antioxidant defense in isolated rat hepatocytes exposed to cadmium.

The aim of the study was the evaluation of cadmium effects on the activity of antioxidant enzymes in rat hepatocytes. The studies were conducted with isolated rat hepatocytes incubated for 1 or 2 hours in a modified (deprived of carbonates with phosphates) Williams' E medium (MWE) in the presence of cadmium chloride (25, 50 and 200 microM). Hepatocytes incubated in the MWE medium without cadmium chloride were used as a control. The application of the modified Williams' E medium allowed for the appearance of cadmium compounds in a soluble form that is indispensable for suitable estimation of its toxic action. There were evaluated markers of the oxidative stress such as: concentration of thiobarbiturate reactive substances (TBARS)--proportional to the level of lipid peroxidation, concentration of reduced glutathione (GSH), and the activity of antioxidant enzymes, including superoxide dismutase (SOD1 and SOD2), catalase (CAT), total glutathione peroxidase (GSHPx), selenium--dependent glutathione peroxidase (SeGSHPx), glutathione transferase (GST) and glutathione reductase (GSHR). Alterations of antioxidant enzymes activity, the level of TBARS and GSH in isolated rat hepatocytes caused by cadmium in vitro, were shown to depend on the concentration and time of exposure of cells to this metal. The increased level of TBARS and GSH was observed as well as changes in the activity of antioxidant enzymes. The activity of SOD isoenzymes and CAT was increased, whereas GSHPx and GST were decreased. These results indicate that cadmium induces oxidative stress followed by alterations in the cellular antioxidant enzyme system in isolated rat hepatocytes.

Hypothermic storage of equine isolated hepatocytes.

The aim of the study was to establish the optimal methods for hypothermic storage of equine isolated hepatocytes. Viability of equine isolated hepatocytes after hypothermic storage was dependent on the type of storage medium as well as on the cell density in the storage suspension and the preservation period. Hepatocytes stored at 4 degrees C in Hanks' Balanced Salt Solution (HBSS) and Williams' Medium E (WE) for 24 h showed very low viability, numerous cell membrane blebs, very low attachment rate (11.9 +/- 6.5% and 34.8 +/- 19.1%, respectively) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction rate (6.4 +/- 3.9% and 25.1 +/- 14.8%, respectively). In contrast, hepatocytes stored in University of Wisconsin Solution (UW) after 24 h of storage at a density of 12.5 x 10(6) cells/ml showed high viability (over 70%), typical and intact morphology, high cell attachment rates and MTT reduction. Our findings clearly demonstrate that UW is a good preservation solution for equine isolated hepatocytes. Hepatocytes harvested from slaughterhouse organs can be stored at 4 degrees C in UW at a density of 12.5 x 10(6) cells/ml for at least 24 h without significant decrease in functional integrity.

Preparation of equine isolated hepatocytes.

In this study a detailed description of the equine hepatocyte isolation procedure is presented. Livers were obtained from horses slaughtered at the local slaughterhouse. For blood removal and liver preservation the following steps are suggested: perfusion with the oxygenated HBSS (0-2 degrees C, with continuous flow of 500-800 ml/min for 3-6 min), protection from ischemia injury by flushing with ice-cold University of Wisconsin Solution (UW, flow rate of 500-800 ml/min), and finally immersion of the liver lobe in UW solution (2 degrees C) during its transport to the laboratory. For equine isolated hepatocyte preparation a "three-step" perfusion procedure was elaborated: rewarming, chelating and collagenase perfusion. We found optimal cell yield and viability under the following conditions: rewarming with UW (38 degrees C) for 8-14 min, chelating with calcium free Hanks' Balanced Salt Solution (HBSS, 38 degrees C) supplemented with 1 mM ethylene glycol-bis[beta-aminoethyl esther]-N,N,N'N'-tetracetic acid at the flow rate of 450 ml/min for 6 min and enzymatic digestion with HBSS supplemented with 0.1% collagenase at 38 degrees C and 450 ml/min flow rate for 8-27 min. These conditions consistently generated cell harvests of 21 x 10(6)+/-4.86 cells/g of perfused liver tissue with viability of 82.7%+/-10.2.

Induction of apoptosis and NF-kappaB by quercetin in growing murine L1210 lymphocytic leukaemic cells potentiated by TNF-alpha.

Polyphenol quercetin induced apoptosis in proliferating murine L1210 lymphocytic cells. DNA damage, as well as apoptosis and withdrawal from the cell cycle were transient. The above mentioned death promoting activity of quercetin was enhanced by physiological concentrations of TNF-alpha. At the same time, indices of cell viability dropped. However, the extent and tendency of the initially enhanced cell mortality steadily diminished throughout the experiment. After 12 h the G2/M phase reappeared. After 24 h all indices almost returned to control levels indicating either the selection of subpopulation of unaffected leukaemic cells or cells developing resistance to the treatment. A DNA ladder of oligonucleosomes was observed for apoptogenic treatments. We conclude that quercetin unmasked cell death, promoting the activity of TNF-alpha. However, after 12 and 24 h of exposure, surviving cells could complete the cell cycle and finally recover. At the same time, increased NF-kappaB activation was demonstrated by immunoblotting of the immunoreactive RelA/p65 subunit in nuclear extracts. Exposure to TNF-alpha or quercetin was crucial for increased activity of NF-kappaB, which may implicate an increasing resistance to their cytotoxicity.

A simple method for hepatocyte attachment in hollow fibre bioreactors.

A new method for hepatocyte attachment in hollow fibre (HF) bioreactors was proposed and verified. A flow of medium with suspended hepatocytes, evoked by transmembrane pressure (TMP), and directed across the membrane into the fibre lumen, has accelerated and improved hepatocyte contact with the HF. It was found that seeding of hepatocytes onto the membrane was optimal at TMP of 50-80 mmHg. Ammonia utilisation and ureagenesis rates in hepatocytes seeded in the bioreactor suggests that the proposed method warrants proper conditions for cell functionality and allows for extended culture of hepatocytes in HF bioreactors. It is speculated that time cutback between introduction of hepatocytes into the bioreactor and the start of the cell attachment process, accomplished by the presented method, leads to substantially improved recovery of freshly isolated hepatocytes, and consequently to better overall performance of HF bioreactor.

Attachment and metabolic activity of hepatocytes cultivated on selected polymeric membranes.

Plasma or hormones added to hepatocyte incubation media mask the function of membranes as substrata per se for hepatocyte adhesion. This hypothesis was verified with hepatocyte cultures on various membranes in serum and hormone free medium. Freshly isolated rat hepatocytes were seeded on flat sheet membranes made of Cellulose Acetate (CA), aminated Cellulose Acetate (aCA), polysulfone (PSf) and sulfonated polysulfone (sPSf) and incubated in Hank's Balanced Salts Solutions (HBSS) as well as in William's E medium supplemented with newborn calf serum. It was found that PSf promoted hepatocyte adhesion most effectively. Good properties of PSf as a biomaterial for hepatocyte culture were confirmed in both media cultures. Urea synthesis and ammonia utilization measured in hepatocytes cultured on PSf were higher compared with other membrane cultures. PSf secured longer viability for a higher number of cells seeded on membrane compared with other investigated membranes, which is the reason for higher metabolic activity in PSf culture.

The fate of nitrogen from 15N-labeled nitrate after single intravenous administration of Na15NO3 in sheep.

The metabolic fate of nitrogen from 15N-labeled sodium nitrate has been investigated in four healthy Polish Merino ewes. 15N-labeled sodium nitrate was administered intravenously at the dosage of 400 micromol.kg(-1) body weight. Blood plasma and urine concentrations of nitrate, ammonia, and urea and 15N enrichment of ammonia and urea were estimated over a 50-h period following 15N-nitrate administration. Nitrate (NO3-) was slowly eliminated from the blood plasma, and the presence of NO3(-) in the blood plasma above the nitrate "background" was observed for 50 h. 15N enrichment of blood plasma urea already appeared at 15 min and reached the maximum 6 h after 15N-nitrate administration. The urinary excretion of nitrate occured during 50 h after 15N-nitrate injection; the total urine excretion of NO3(-) was 23.63+/-2.39% of the administered dose. The mean urinary recoveries of nitrogen as 15N-urea and 15N-ammonia were 14.76+/-1.32% and 0.096+/-0.015% of the administered 15N-nitrate dose, respectively. It should be pointed out that in total only 38.49% of the administered nitrate-N was excreted in urine (as nitrate, ammonia and urea nitrogen) during 50 h. The results obtained indicate that sheep are able to store nitrate nitrogen in their body. The fate of the remaining approximately 60% of the 15NO3(-) administered dose is unknown. The results obtained do not allow one to conclude what fraction of the unrecovered approximately 60% of the 15NO3(-) dose was utilized by gastrointestinal microorganisms, and (or) metabolized, or stored in sheep tissues.

In vitro ammonia utilization and urea synthesis by rat liver after portacaval shunt.

BACKGROUND: The objective of this study was to study in vitro ammonia utilization and urea synthesis by the rat liver after portacaval shunt (PCS) in comparison to controls. This study is part of a series of experiments to investigate metabolic derangements in the liver following PCS. METHODS: Sixteen male Wistar rats weighing 150-230 g were used. PCS was performed on 8 and the rest served as sham-operated controls. The experiments were performed 4 weeks postoperatively. The usually monitored postoperative parameters after PCS like body weight, rise in ammonia levels in the peripheral blood, atrophy of mesenteric fat, atrophy of the liver, etc., were comparable to other PCS series. Liver slices (1 x 2 x 1 mm) were prepared from each rat liver and incubated in Krebs-Henseleit buffer. RESULTS: After incubation, ammonia utilization in the controls was 162.88+/-20.12 micromol/g dry weight/h while in PCS rats it was 81.35+/-2.64 micromol/g dry weight/h (p < 0.0001). The rate of urea synthesis in controls was 122.54+/-12.93 micromol/g dry weight/h whereas in the PCS group it was 14.99+/-2.21 micromol/g dry weight/h (p < 0.0001). CONCLUSION: These results indicate a significant decline in the capacity of the liver for ammonia utilization as well as urea synthesis after PCS.

The influence of nitrite, nitrate and nitric oxide on ammonia use and urea production in primary cultures of sheep hepatocytes.

Inorganic nitrite introduced into the living organism is rapidly converted into nitrate and nitric oxide (NO). It is known that nitrite decreases ammonia use and urea formation in vitro and it seems that nitrite also influences these processes in vivo. However, the mechanism underlying this effect is not known. Therefore, we decided to compare the influence of sodium nitrite (NaNO(2)), sodium nitrate (NaNO(3)) and NO on ammonia use and ureagenesis in monolayer cultures of sheep hepatocytes during 18 hr of cultivation. It was found that 0.5 and 2.5 mmNaNO(2) significantly reduced ammonia use in a dose-dependent manner for the first 6 hr of incubation; at higher concentrations (2.5 mm), it also decreased urea formation. Also, the presence of nitrite did not affect the lactate dehydrogenase (LDH) activity in the medium which indicates that the nitrite effect did not result from its cytotoxic action. NaNO(3) (0.5 and 2.5 mm) did not induce any changes in ammonia use and urea synthesis in hepatocytes. With sodium nitroprusside (SNP) (0.001, 0.01, 0.1, 0.5 and 1.0 mm) a decrease of ammonia use and urea production was observed corresponding to the nitrite effect, but contrary to nitrite exposure these changes in metabolism were persistent during the whole cultivation period. On the other hand, potassium cyanide (KCN) (0.1 and 0.5 mm) did not influence either urea formation or ammonia use. Thus, it can be concluded that in isolated hepatocytes nitrate and/or NO are not the mediators of nitrite effects on nitrogen metabolism. Furthermore, there is no evidence that nitrite effects are mediated by impaired mitochondrial respiration and energy production.

Effect of nitrite on ureagenesis and carbohydrate metabolism in isolated rat hepatocytes.

The effect of three NaNO2 concentrations (0.5, 2.0, and 5.0 mM) on 15N-ammonia utilization, ureagenesis, glucose, pyruvate and lactate formation and glycogen breakdown were studied in isolated rat hepatocytes. Nitrite failed to affect the rate of glycogenolysis as well as the lactate and pyruvate formation, but at the same time it markedly increased the glucose formation. It is concluded that the increase in the glucose formation results from the nitrite stimulation of the rate of gluconeogenesis. An increased sodium nitrite concentration caused a significant decrease in the ammonia utilization and urea synthesis; there are strong linear correlations between the nitrite concentration and the amount of utilized ammonia (r = -0.93) and the formed urea (r = -0.96). The observed lower rate of ureagenesis in the presence of nitrite resulted from the diminished incorporation of the added 15N-ammonia into urea, as well as from the diminished urea formation from endogenous nitrogen. It is concluded that the disturbances in carbohydrate and nitrogen metabolism observed in the nitrite-poisoned animals are attributed to the direct effect of nitrite on metabolism.

Effect of propionate on the utilization of nitrogen from 15NH4Cl for urea synthesis in hepatocytes isolated from sheep liver.

1. The effect of ornithine (2.0 mM) and propionate (5.0 mM) on the utilization of N from 15NH4Cl (5.0 mM) for urea synthesis in hepatocytes isolated from sheep liver was investigated. 2. The capacity of sheep hepatocytes to utilize [15N]ammonia in the absence of the other exogenous substrates was very low and amounted 132 +/- 37.3 mumol/hr per 1 g dry wt. 3. Ornithine failed to affect the total [15N]ammonia uptake and total urea synthesis, but at the same time it markedly increased the utilization of [15N]ammonia for ureagenesis and diminished the rate of urea synthesis from endogenous sources. 4. Propionate markedly increased total [15N]ammonia utilization and total urea formation; this increase resulted from the rise of ammonia utilization for urea synthesis and it was similar in the presence or absence of ornithine. 5. The capacity of sheep liver cells to utilize ammonia in the presence of propionate (in the presence or absence of ornithine) amounted to 256 mumol/hr per 1 g dry wt, thus being similar to the values in vivo. 6. It is concluded that in sheep hepatocytes both ornithine and propionate stimulate the utilization of ammonia for urea synthesis and these effects take place independently and occur by different mechanisms.