Efficacy of trimethoprim-sulfadoxine against Escherichia coli in a tissue cage model in calves.

Tissue cages implanted subcutaneously in calves were infected with Escherichia coli. Twenty-four hours later, the calves were treated either with single doses of 2.5 + 12.5 or 5 + 25 mg/kg trimethoprim (TMP) + sulfadoxine (SDX) or with five doses of 7.5 + 37.5 mg/kg TMP + SDX at 12-h intervals. In addition, one cage in each of three calves in the highest dose group was infected 3 h after initiation of treatment. Untreated calves were kept as controls. Concentrations of TMP and SDX in plasma and tissue cage fluid (TCF) and counts of viable bacteria in TCF were determined. In the highest dose group, concentrations of TMP in TCF remained above the minimum inhibitory concentration of the test strain for 94-101 h and peak to minimum inhibitory concentration (MIC) ratio was close to 10. In spite of this, an effect of treatment was noted only in cages infected after initiation of treatment. In vitro studies and analysis of thymidine content in serum and TCF from calves suggest that levels of thymidine in TCF are high enough to antagonize the antibacterial effect of TMP. The results indicate that soft tissue infections in secluded infection sites of calves are refractory to treatment with TMP + SDX.

Cellular transport of anandamide, 2-arachidonoylglycerol and palmitoylethanolamide--targets for drug development?

The endogenous cannabinoid anandamide (AEA) is transported into cells by a temperature-sensitive process of facilitated diffusion. This uptake process has been characterised both biochemically and pharmacologically, and shown to be regulated at least in part by the intracellular metabolism of the accumulated AEA by fatty acid amide hydrolase. In this review, the properties of this transport process are briefly reviewed together with the corresponding transport mechanisms for the related endogenous compounds 2-arachidonoylglycerol and palmitoylethanolamide. In addition, the possibility that these transport mechanisms can be targets for therapeutic strategies aimed at prolonging the effects of the endocannabinoids is discussed.

Inhibition of rat C6 glioma cell proliferation by endogenous and synthetic cannabinoids. Relative involvement of cannabinoid and vanilloid receptors.

The effects of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) upon rat C6 glioma cell proliferation were examined and compared with a series of synthetic cannabinoids and related compounds. Cells were treated with the compounds each day and cell proliferation was monitored for up to 5 days of exposure. AEA time- and concentration-dependently inhibited C6 cell proliferation. After 4 days of treatment, AEA and 2-AG inhibited C6 cell proliferation with similar potencies (IC(50) values of 1.6 and 1.8 microM, respectively), whereas palmitoylethanolamide showed no significant antiproliferative effects at concentrations up to 10 microM. The antiproliferative effects of both AEA and 2-AG were blocked completely by a combination of antagonists at cannabinoid receptors (SR141716A and SR144528 or AM251 and AM630) and vanilloid receptors (capsazepine) as well as by alpha-tocopherol (0.1 and 10 microM), and reduced by calpeptin (10 microM) and fumonisin B(1) (10 microM), but not by L-cycloserine (1 and 100 microM). CP 55,940, JW015, olvanil, and arachidonoyl-serotonin were all found to affect C6 glioma cell proliferation (IC(50) values of 5.6, 3.2, 5.5, and 1.6 microM, respectively), but the inhibition could not be blocked by cannabinoid + vanilloid receptor antagonists. It is concluded that the antiproliferative effects of the endocannabinoids upon C6 cells are brought about by a mechanism involving combined activation of both vanilloid receptors and to a lesser extent cannabinoid receptors, and leading to oxidative stress and calpain activation. However, there is at present no obvious universal mechanism whereby plant-derived, synthetic, and endogenous cannabinoids affect cell viability and proliferation.

Effects of the cannabimimetic fatty acid derivatives 2-arachidonoylglycerol, anandamide, palmitoylethanolamide and methanandamide upon IgE-dependent antigen-induced beta-hexosaminidase, serotonin and TNF alpha release from rat RBL-2H3 basophilic leukaemic cells.

There are conflicting reports in the literature as to whether palmitoylethanolamide affects the function of mast cell-related cell lines in vitro, in contrast to the well-documented effects of this compound upon mast cell function in vivo. In the present study, we have reinvestigated the effects of palmitoylethanolamide upon antigen-induced release of [3H]serotonin and beta-hexosaminidase from rat basophilic leukemia RBL-2H3 cells and compared these effects with those of 2-arachidonoylglycerol, anandamide and R1-methanandamide. RBL-2H3 cells were sensitized with a monoclonal anti-DNP IgE, after which they were stimulated with antigen (DNP-HSA). Palmitoylethanolamide produced a small, but significant reduction in antigen-stimulated [3H]serotonin release at high concentrations, whereas anandamide was without effect. In contrast, 2-arachidonoylglycerol and methanandamide increased the antigen-stimulated release of both [3H]serotonin and beta-hexosaminidase. It is concluded that in RBL-2H3 cells, these cannabimimetic fatty acid derivatives do not have potent stabilizing effects upon antigen-induced degranulation.

Pharmacological properties of cannabinoid receptors in the avian brain: similarity of rat and chicken cannabinoid1 receptor recognition sites and expression of cannabinoid2 receptor-like immunoreactivity in the embryonic chick brain.

The pharmacological properties of brain cannabinoid receptors were investigated in brains of 35 day-old chickens, since little is known about the avian cannabinoid system. The cannabinoid1 receptor-selective antagonist ligand [3H]SR 141716A bound to chicken brain membranes with K(D) and Bmax values of 0.92+/-0.28 nM and 790+/-58 fmol/mg protein, respectively. The binding was inhibited by CP 55,940 with a pI50 value of 7.63+/-0.14 and by a series of compounds with the order of potency CP 55,940>R(+)WIN 55,212-2>R-1 methanandamide approximately DAK. S(-)WIN 55,212-3 and AM404 were without inhibitory effect at 1 microM. Similar results were found for rat brain membranes. For both rat and chicken brain membranes, addition of the non-hydrolysable GTP analogues Gpp[NH]p and GTPgammaS shifted the CP 55,940 inhibition curve to the right, consistent with an intact coupling to G-proteins in the preparations. Fatty acid amidohydrolase in chicken brain membranes was less sensitive to inhibition by phenylmethylsulphonyl fluoride and arachidonoyl serotonin than its rodent equivalent. However, when fatty acid amidohydrolase activity in the preparations was reduced by use of a lower assay membrane concentration, anandamide was found to inhibit the binding of [3H]SR 141716A to chicken membranes with a pI50 value of 6.39+/-0.16. Using a novel antibody raised to amino acids 346-359 from the C-terminal tail of the human cannabinoid2 receptor, it was found that embryonic chick brain tissue (and embryonic chick neurones in primary culture) expressed a approximately 53 kDa immunoreactive band. This immunoreactivity, which was prevented by preincubation of the antibody with the immunising peptide, was also seen in cells expressing the recombinant human cannabinoid, receptor, but was not seen in adult chicken brain homogenates or in rat cerebellar homogenates. However, a "classical" cannabinoid2-receptor component of [3H]WIN 55212-2 binding (i.e. a fraction inhibited by low concentrations of the cannabinoid2-receptor-selective antagonist SR 144528) was not found.

Characterization of palmitoylethanolamide transport in mouse Neuro-2a neuroblastoma and rat RBL-2H3 basophilic leukaemia cells: comparison with anandamide.

The endogenous cannabinoid receptor agonist anandamide (AEA) and the related compound palmitoylethanolamide (PEA) are inactivated by transport into cells followed by metabolism by fatty acid amide hydrolase (FAAH). The cellular uptake of AEA has been characterized in detail, whereas less is known about the properties of the PEA uptake, in particular in neuronal cells. In the present study, the pharmacological and functional properties of PEA and AEA uptake have been investigated in mouse Neuro-2a neuroblastoma and, for comparison, in rat RBL-2H3 basophilic leukaemia cells. Saturable uptake of PEA and AEA into both cell lines were demonstrated with apparent K(M) values of 28 microM (PEA) and 10 microM (AEA) in Neuro-2a cells, and 30 microM (PEA) and 9.3 microM (AEA) in RBL-2H3 cells. Both PEA and AEA uptake showed temperature-dependence but only the AEA uptake was sensitive to treatment with Pronase and phenylmethylsulfonyl fluoride. The AEA uptake was inhibited by AM404, 2-arachidonoylglycerol (2-AG), R1- and S1-methanandamide, arachidonic acid and olvanil with similar potencies for the two cell types. PEA, up to a concentration of 100 microM, did not affect AEA uptake in either cell line. AEA, 2-AG, arachidonic acid, R1-methanandamide, (9)-THC, and cannabidiol inhibited PEA transport in both cell lines. The non-steroidal anti-inflammatory drug indomethacin inhibited the AEA uptake but had very weak effects on the uptake of PEA. From these data, it can be concluded that PEA is transported in to cells both by passive diffusion and by a facilitated transport that is pharmacologically distinguishable from AEA uptake.

Serum-dependent effects of tamoxifen and cannabinoids upon C6 glioma cell viability.

In the present study, the effects of the combination of tamoxifen ((Z)-2[p-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylamine citrate) and three cannabinoids (Delta(9)-tetrahydrocannabinol [Delta(9)-THC], cannabidiol, and anandamide [AEA]) upon the viability of C6 rat glioma cells was assessed at different incubation times and using different culturing concentrations of foetal bovine serum (FBS). Consistent with previous data for human glioblastoma cells, the tamoxifen sensitivity of the cells was increased as the FBS content of the culture medium was reduced from 10 to 0.4 and 0%. The cells expressed protein kinase C alpha and calmodulin (the concentration of which did not change significantly as the FBS concentration was reduced), but did not express estrogen receptors. Delta(9)-THC and cannabidiol, but not AEA, produced a modest reduction in cell viability after 6 days of incubation in serum-free medium, whereas no effects were seen in 10% FBS-containing medium. There was no observed synergy between the effects of tamoxifen and the cannabinoids upon cell viability.

Neurotoxicity of glutamate in chick telencephalon neurons: reduction of toxicity by preincubation with carbachol, but not by the endogenous fatty acid amides anandamide and palmitoylethanolamide.

Exposure of chick telencephalon neurons in serum-free primary culture to glutamate produced a concentration-dependent cell toxicity as seen by an increase in lactate dehydrogenase (LDH) release that was blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine and was reduced by preincubation with the cholinergic agonist carbachol. Preincubation with a threshold concentration of NMDA did not prevent glutamate toxicity, suggesting that chick NMDA receptors do not desensitize in the manner reported for their rodent counterparts. Neither anandamide (arachidonyl ethanolamide, AEA) nor palmitoylethanolamide (PEA) was able to prevent the neurotoxicity produced by prolonged glutamate incubation, even under conditions in which the metabolism of the compounds by fatty acid amide hydrolase or AEA cellular uptake was blocked. It is concluded that treatments reported as granting neuroprotection towards glutamate toxicity in rodent primary neuronal cultures do not necessarily show the same properties in the chick.

Investigation into the effects of amyloid (1-42) beta-peptide upon basal and antigen-stimulated hexosaminidase and serotonin release from rat RBL-2H3 basophilic leukemia cells.

There is evidence that beta-amyloid (A beta) peptide infusion in vivo produces a degranulation of vascular mast cells. It would be useful to investigate the interaction between A beta and mast cells in a simple in vitro model system in order to determine the cellular mechanism by which exposure to A beta peptides results in mast cell degranulation. In the present study, the effect of A beta(1-42) upon the release of granular hexosaminidase and serotonin has been investigated using the cognate rat mast cell line, RBL-2H3. Sensitization of these cells for 1 h with anti-DNP IgE (monoclonal anti-dinitrophenyl) results in a large release of hexosaminidase and serotonin due to degranulation when the cells are exposed to DNP-HSA (albumin, human dinitrophenyl). Pretreatment overnight with A beta(1-42) (10 and 30 microM) did not affect either the basal or antigen-stimulated release of hexosaminidase or serotonin. A similar lack of effect of A beta(25-35) and the lipid peroxidation product HNE upon antigen-stimulated release of hexosaminidase or serotonin was also found. It is concluded that RBL-2H3 cells are not a useful model for mechanistic studies into the effects of beta-amyloid peptides upon vascular mast cells.

Increased levels of nitrogen oxides and lipid peroxidation in the rat brain after soman-induced seizures.

We have investigated the effect of soman-induced seizures on rat brain levels of nitrogen oxides (NOx) and lipid peroxidation (LPO) 30 min and 24 h after intoxication. Following administration of soman (90 microg/kg s.c.), acetylcholinesterase activity was reduced to <10% of control after 30 min, whereas some de novo synthesis had occurred after 24 h. Significant increases in the LPO products malondialdehyde (MDA) and (E)-4-hydroxy-2-nonenal (4-HNE) were seen in the cortex, hippocampus, striatum, thalamus and medulla-pons 30 min after administration. A significant increase in the brain NOx levels, suggesting an increase in NO production, was seen in the cortex after 30 min and in the hippocampus and the striatum after 24 h. No significant changes were observed in cerebellum. These data suggest the possibility that free radical reactions may be a primary cause of neuronal degeneration after soman intoxication.

The effect of hydrogen peroxide upon beta-adrenoceptor density and function in C6 rat glioma cells.

Hydrogen peroxide has been suggested to play an important role in the pathogenesis of Alzheimer's disease. In the present study, the effects of hydrogen peroxide upon the functional integrity of beta-adrenoceptors have been investigated in C6 glioma cells. Treatment of cells for 24 h with hydrogen peroxide in serum-free medium produced a concentration-dependent cell toxicity, seen both using cell counting and LDH release into medium as end point. There were no large nor consistent changes in either the density of cell surface beta 1, or beta 2-adrenoceptors, measured using the hydrophilic ligand [3H](-)-CGP 12177, nor in either basal, forskolin and isoprenaline-stimulated cAMP responses, following hydrogen peroxide treatment. It is concluded that the decreased adenylyl cyclase activity and responsiveness to Gs stimulation found in post-mortem brain samples from Alzheimer's disease autopsy cases is unlikely to be mediated by hydrogen peroxide.

Dopamine and glutamate neurotoxicity in cultured chick telencephali cells: effects of NMDA antagonists, antioxidants and MAO inhibitors.

In a recent study, it was found that the intrastriatal administration to rats of the organophosphorous compound soman and kainic acid produced a rapid release not only of glutamate but also of dopamine in this brain region. Dopamine is a potent source of free radicals and is known to produce cytotoxic effects, per se. This raises the possibility that the released glutamate and dopamine act synergistically to produce the neurotoxicity found after soman administration. In order to investigate the feasibility of this hypothesis in an in vitro system, the effects of dopamine and glutamate upon cell survival were investigated using chick neurons (7 DIV) in serum-free primary culture. The neurons were treated with dopamine and/or glutamate for up to 24 h and cell toxicity was then assessed either by determination of cell densities, by the release of cytoplasmic LDH or by the MTT cytotoxicity assay. L-Glutamate produced a concentration-dependent cytotoxicity that was seen as early as after 30 min of exposure, and was accompanied by an increased level of lipid peroxidation. The L-glutamate toxicity could to a large extent by prevented by NMDA receptor antagonists and to a lesser extent by catalase, superoxide dismutase or glutathione ethyl ester added 30 min before the glutamate. Dopamine was also cytotoxic, and the cytotoxicity was reduced by the combination of catalase and glutathione ethyl ester but not by the MAO inhibitors clorgyline or L-deprenyl, or by the selective dopamine uptake inhibitor GBR 12783. The cytotoxic effects of dopamine and L-glutamate were additive rather than synergistic, regardless of the incubation time used. It is concluded that chick neurons in serum-free culture are a useful in vitro model system for the study of cell toxicity produced by oxidative stress and by glutamate. The cytotoxic effects of dopamine in this model are not due to the monoamine oxidase-mediated production of hydrogen peroxide but appear at least in part to be related to oxidative stress.

Flumethasone-induced insulin resistance in calves.

The impact of one single therapeutic dose of flumethasone (0.5 mg/100 kg b.w.) on insulin sensitivity was studied in calves. Hyperinsulinemic, euglycemic clamp tests were performed before and after flumethasone treatment. At 24 h after treatment, insulin-dependent glucose utilization was reduced by 74% (P < 0.005). No significant changes occurred 72 h post-treatment.

Clenbuterol-induced insulin resistance in calves measured by hyperinsulinemic, euglycemic clamp technique.

Hyperinsulinemic, euglycemic clamp tests were performed on calves before and after clenbuterol treatment. Clenbuterol was given as 2 intramuscular injections with an interval of about 12 h. The dose used was 1 microgram/kg b.w. The treatment resulted in increased plasma levels of insulin and glucose. The results of the clamp tests showed that clenbuterol induced a transient decrease in insulin sensitivity. Both insulin mediated glucose disposal (M), expressed as mumol/kg live b.w./min. and the M/I-index (M divided by the average insulin concentration at steady state) were significantly reduced after treatment. The effect of clenbuterol on carbohydrate metabolism seemed to be rather short-lived, since significant changes occurred only in animals treated 5-6 h prior to the test. According to the literature, the metabolic effects of clenbuterol have been studied only after the high doses used for growth promoting purposes. The results from the present study showed that similar changes occur also after doses at the therapeutic level. The hyperinsulinemic, euglycemic clamp test was considered to be a valuable tool for the study of insulin sensitivity in cattle.

Effects of intravenous infusion of glucose and pancreatic glucagon on abomasal function in dairy cows.

Four cows of the Swedish red and white breed fitted with a cannula in the abomasum were used in 2 experiments. In experiment I glucose (4 mg/kg bw/min) was infused intravenously for 60 min after an initial control period, without infusion, of 60 min. The turnover time of abomasal fluid was calculated using Cobalt-EDTA as fluid marker. The frequency and amplitude of the abomasal pressure changes were registered during the experiment. The plasma level of insulin and glucose was also registered during the experiment. Due to the glucose infusion plasma glucose increased with about 4 mmol/l. The elevated plasma level of glucose induced a pronounced release of insulin. The turnover time of abomasal fluid increased from 15.7 +/- 1.2 to 27.8 +/- 3.5 min (p < 0.01) during the glucose infusion. The mean amplitude of the pressure changes showed a more than twofold increase (p < 0.05) during glucose infusion as compared with the control period but there was no difference in the frequency of the changes. In experiment II there was a similar experimental set-up with the exception that pancreatic glucagon (30 pg/kg bw/min) was infused instead of glucose. The glucagon infusion induced a release of endogenous glucose which in turn increased the plasma level with about 3 mmol/l. The plasma level of insulin rose to about the same extent as during the glucose infusion in experiment 1. The turnover time of abomasal fluid was delayed from 15.4 +/- 1.7 to 34.8 +/- 1.9 min (p < 0.001). There were no significant effects of the glucagon infusion on the frequency or the amplitude of the abomasal pressure changes. The results of the present study indicate a disturbed abomasal function in cattle with hyperglycaemia. It remains to be investigated if it is a direct effect of the hyperglycaemia or if it is secondary to the elevated insulin level.

Characterization of the N-methyl-D-aspartate (NMDA) receptor in the embryonic chick brain.

Relatively little is known about the properties of the NMDA receptor in avian species. In the present study, NMDA receptor pharmacology and function has been studied in the embryonic chick brain. The competitive antagonist ligand [3H]CGP 39653 bound to telencephali membranes from 17-day-old chick embryos with KD and Bmax values of 6.6 nM and 3.3 pmol/mg protein, respectively. The binding was inhibited by CGS 19755, L-glutamate, R-CPP and D-AP5 with pI50 values of 7.57, 7.49, 7.28 and 7.08, respectively. L-glycine, S-AMPA, kainate and MK-801 only weakly affected the binding. The inhibitory potency of NMDA (pI50 value 6.98), however, was greater than seen in other species, suggesting a species difference in receptor structure. Such a possibility was investigated by use of two NMDA-R1 antibodies, AB 1516 and 69921 A, which recognize the C-terminal region and an intracellular domain of the rat receptor, respectively. Immunoreactive signals (approximately 116 kDa) were found for a rat brain homogenate, whereas only 69921A gave an immunoreactive signal in the chick. The functional status of the NMDA receptors was investigated in serum-free cultures of neurons from 8-day-old chick embryos. Glutamate and NMDA were found concentration-dependently, and in a manner that could be antagonized by CGS 19755 and MK-801, to produce cell death. Thus, the present study indicates that chick embryonic brain expresses functional NMDA receptors that are pharmacologically and possibly structurally different from their rodent counterparts.

Pharmacokinetics of penicillin-G in ewes and cows in late pregnancy and in early lactation.

Concentrations of penicillin-G in serum were determined after single intravenous doses of potassium penicillin-G (10 mg/kg body wt) to ewes and cows in late pregnancy and in early lactation. Penicillin-G in serum was analysed by a microbiological method and pharmacokinetic parameters were calculated by model-independent methods. Serum concentrations were lower in early lactation than in late pregnancy in both ewes and cows. The differences were statistically significant at most sampling times in ewes but only during the first hour in cows. Weight corrected values of clearance and volume of distribution were significantly higher in early lactation than in late pregnancy in both ewes and cows. Mean residence time and elimination-half life were shorter in lactating than in pregnant ewes, however for the latter parameter the difference was not statistically significant. Neither of these parameters changed in cows. It is concluded that in both ewes and cows pharmacokinetic parameters of penicillin-G are altered from late pregnancy to early lactation but that these changes have little practical impact and do not call for a revised dosage regime of the studied drug.

Correlation between cortical EEG and striatal microdialysis in soman-intoxicated rats.

In vivo microdialysis and EEG recording have been used in order to study the combined neurochemical and electrophysiological events during intoxication with soman (o-1,2,2-trimethylpropyl methylphosphono-fluoridate), a potent inhibitor of acetylcholinesterase (AChE), in the freely moving rat. All rats exposed to soman exhibited signs of AChE inhibition. The duration of EEG recorded seizures after soman intoxication averaged 43 +/- 24 min. The extracellular striatal levels of dopamine and GABA, increased significantly during the EEG seizure periods. Using an EEG based differentiation between seizure and non-seizure conditions, we found that intrastriatal release of dopamine, but not glutamate, during soman intoxication is highly correlated with seizures. Our results suggest that excitatory amino acids (EAA) involvement in soman-induced seizures, as demonstrated in hippocampus, may not be relevant in the striatum. Our data, instead, may indicate the importance of dopamine as a neurotoxic agent.

Release of dopamine, GABA and EAA in rats during intrastriatal perfusion with kainic acid, NMDA and soman: a comparative microdialysis study.

There is an increasing amount of experimental evidence that excitatory amino acids (EAAs) are involved in the brain lesions observed after severe intoxication with the highly toxic organophosphorus compound soman. This study was undertaken to compare the acute actions of soman, and the glutamatergic receptor agonists kainic acid and N-methyl-D-aspartate (NMDA) on striatal release of dopamine and amino acids. The neurotoxic compounds were administered in high (10 mM) concentrations by unilateral intrastriatal microdialysis perfusion in freely moving rats. During the microdialysis the animals were observed for toxic signs related to convulsion. The glial fibrillary acidic protein (GFAP) was monitored as a marker of neurotoxicity in parts of prefrontal cortex, hippocampus, striatum and cerebellum. Acetylcholinesterase (AChE) inhibition in six brain regions was measured after soman perfusion in order to assess its cerebral distribution. We found that soman perfusion induced a major release of dopamine, GABA and aspartate in the striatum. Kainic acid also induced a release of dopamine and aspartate. NMDA was not as potent an inducer of striatal neurotransmitter release as soman and kainic acid. Soman and kainic acid perfusion produced convulsive behaviour in the rats. The main neurochemical event in the striatum during soman- and kainate-induced convulsions is the release of dopamine. We suggest that this major dopamine release might be as important as an increase in EAA in the cascade of pathological events leading to the brain damage in the striatum observed after soman intoxication.