Fritz Rupert Ungemach

Oral bioavailability of quercetin from different quercetin glycosides in dogs

Although the flavonol quercetin is used as a supplement in commercial dog food, data on quercetin bioavailability in dogs are not available. Thus, we investigated quercetin bioavailability (measured as area under the concentration-time curve) in nine adult beagle dogs at an oral dose of 10 mg/kg body weight (b.w.). The major fraction (>80 %) of flavonols circulating in blood plasma were conjugated metabolites of quercetin. The absolute bioavailability of quercetin (i.e. the fraction that reaches the systemic circulation) was only about 4 %. We also compared the oral bioavailability between the aglycone quercetin and its more often used glucorhamnoside (rutin) and 3-O-glucoside (isoquercitrin) at an equimolar dose of 30 mumol/kg b.w. (corresponding to 10 mg quercetin/kg). Quercetin and isoquercitrin were mainly absorbed in the small intestine with isoquercitrin being one and a half times more bioavailable than quercetin. Maximal plasma concentration after isoquercitrin treatment was 0.89 (sem 0.07) mumol/l. Although quercetin absorption from rutin was delayed, relative bioavailability was not lower than from the aglycone itself. The latter observation is in clear contrast to findings in human subjects, pigs or rats and might indicate that rutin is a better source of quercetin in dogs than in other species. However, potential in vivo quercetin effects beyond the gastrointestinal tract are limited by the intensive metabolism as well as by the rather low bioavailability of this flavonol.

Formation of 4-hydroxynonenal and further aldehydic mediators of inflammation during bromotrichlorornethane treatment of rat liver cells

Bromotrichloromethane (CBrCl3) treatment is a model for studies on molecular mechanisms of haloalkane toxicity with some advantages compared with CCl4 treatment. The formation of 4-hydroxynonenal and similar aldehydic products of lipid peroxidation, which play a role as mediators of inflammatory processes, was clearly demonstrated in rat hepatocytes treated with CBrCl3. It may be assumed that haloalkane toxicity is connected with the biological effects of those inflammation mediatory aldehydic compounds.

Pharmacological and biochemical characterization of the beta-adrenergic signal transduction pathway in different segments of the respiratory tract.

Although in the respiratory system there is great therapeutic interest in manipulating and understanding the beta-adrenoceptor-G-protein-adenylate cyclase (AC) signal transduction pathway, little is known on segmental differences among lung, bronchus, and trachea with regard to the receptor concentration and interaction to G-proteins and coupling to AC. In this study, patterns of distribution and absolute quantities of beta-adrenoceptor subtypes beta(1) and beta(2) were determined in membranes of equine lung parenchyma, bronchial and tracheal epithelium with the underlying smooth muscle by saturation and competition binding assays using the radioligand (-)-[125I]-iodocyanopindolol (ICYP). Additionally, the functional coupling of beta-adrenoceptors to G-proteins (assessed by beta-agonist competition binding in the presence and absence of GTP) as well as the coupling efficiency and biochemical activities of AC was investigated in each region. The specific ICYP binding was rapid, reversible, saturable with time and of high affinity. The radioligand binding identified more total beta-adrenoceptors in the lung than in bronchus or trachea (428+/-19, 162.4+/-4.8, 75.6+/-1.2 fmol/mg protein, respectively) with about 40% of receptors in the high affinity state. The beta(2)-adrenoceptor subtype predominated in all segments (approximately 74-80%), as the highly selective beta(2)-adrenoceptor antagonist ICI 118,551 was about 10,000 times more potent in inhibiting ICYP binding than was the beta(1)-selective adrenoceptor antagonist CGP 20712A, and beta-adrenoceptor agonists inhibited ICYP binding with an order of potency: (-)-isoprenaline>(-)-adrenaline>(-)-noradrenaline. The dissociation constant (K(d)) was higher in the trachea than in bronchus or lung (13.0+/-0.9 pM vs. 20.0+/-2.3 pM vs. 30.8+/-4.4 pM, P<0.05, respectively). The beta(2)-adrenoceptor-mediated AC response was tissue-dependent; stimulants acting on beta-adrenoceptor (isoproterenol), G-protein (GTP, NaF) and AC (forskolin, Mn(2+)) enhanced AC responses in all three regions, but the AC activity was higher in tracheal crude membranes than in bronchus or lung (trachea>>>bronchus>lung), hence, the number of beta(2)-adrenoceptors correlated inversely with the amount of AC. We conclude that (1) the stoichiometry of components within the pulmonary beta-adrenoceptor-G-protein complex is segment-dependent, and (2) the receptor number or AC activity is possibly the rate-limiting factor in the beta-adrenoceptor-G-protein-AC-mediated physiological responses. Thus, it is speculated that this could have important therapeutic consequences in beta-adrenoceptor agonist-induced receptor regulation in bronchial asthma.

Possible Role of Dexamethasone in Sensitizing the Beta-2-Adrenergic Receptor System in vivo in Calves during Concomitant Treatment with Clenbuterol

β2-Agonists blunt the function of the β-adrenoceptor G-protein adenylate cyclase-signalling system, whereas glucocorticoids reverse the agonist-mediated diminished β-adrenergic responses; however, these effects have not been reported in vivo in calf lymphocytes. In this study, we first investigated the presence of the β2-adrenergic receptors on calf lymphocytes, and second we tested the effects of either clenbuterol alone or in combination with dexamethasone on receptor expression and function (isoproterenol-induced intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) formation) in vivo. (–)-[125I]-Iodocyanopindolol (ICYP) binding to intact calf lymphocytes was rapid, saturable (maximal number of binding sites 987 ± 89 ICYP-binding sites/cell, n = 4) and of high affinity (KD value 17.23 ± 2.8 pmol/l, n = 4). These binding sites were of the β2-subtypes of adrenoceptors as indicated by the fact that β-agonists inhibited ICYP binding with an order of potency: (–)-isoproterenol > (–)-adrenaline > (–)-noradrenaline. Furthermore, the selective β2-adrenoceptor antagonist ICI 118.551 was about >1,500 times more potent in inhibiting ICYP binding than was the β1-selective adrenoceptor antagonist CGP 20712A. Consequently, calves were treated with clenbuterol (1.0 µg/kg b.i.d., i.v.) for 9 days alone or simultaneously with dexamethasone (0.1 mg/kg, i.v., once a day for 4 days). Clenbuterol decreased the number of lymphocyte β2-adrenergic receptors by about 40–50% after only 48 h of drug administration. This was accompanied by a decrement in isoproterenol-induced lymphocyte cAMP formation. Upon application of both drugs, dexamethasone restored the clenbuterol-mediated decrease in β2-adrenoceptors and cAMP production. Dexamethasone elevated the number of β2-adrenoceptors and cAMP almost 1.5- to 2-fold at 24 h of drug administration, an effect that persisted for up to 24 h following drug withdrawal. Neither clenbuterol nor the combination with dexamethasone had an influence on the affinity of the receptor for the ligand. The present results demonstrate that dexamethasone in vivo upregulates the number and function of calf lymphocyte β2-adrenoceptors, and thus enhances the sensitivity of the β2-adrenoceptor signal-transduction pathway for clenbuterol during concomitant treatment with both drugs.

Growth and differentiation of primary and passaged equine bronchial epithelial cells under conventional and air-liquid-interface culture conditions

BackgroundHorses develop recurrent airway obstruction (RAO) that resembles human bronchial asthma. Differentiated primary equine bronchial epithelial cells (EBEC) in culture that closely mimic the airway cells in vivo would be useful to investigate the contribution of bronchial epithelium in inflammation of airway diseases. However, because isolation and characterization of EBEC cultures has been limited, we modified and optimized techniques of generating and culturing EBECs from healthy horses to mimic in vivo conditions.ResultsLarge numbers of EBEC were obtained by trypsin digestion and successfully grown for up to 2 passages with or without serum. However, serum or ultroser G proved to be essential for EBEC differentiation on membrane inserts at ALI. A pseudo-stratified muco-ciliary epithelium with basal cells was observed at differentiation. Further, transepithelial resistance (TEER) was more consistent and higher in P1 cultures compared to P0 cultures while ciliation was delayed in P1 cultures.ConclusionsThis study provides an efficient method for obtaining a high-yield of EBECs and for generating highly differentiated cultures. These EBEC cultures can be used to study the formation of tight junction or to identify epithelial-derived inflammatory factors that contribute to lung diseases such as asthma.

Dose-dependent emetic effects of the Amaryllidaceous alkaloid lycorine in beagle dogs

Ingestions of plant material from Amaryllidaceae, especially the bulbs of daffodils, are known to be toxic, representing a persistent cause of poisoning in human and animals. Empiric data from case reports suggested, that the alkaloid lycorine could be the toxic constituent of the multi-component mixture responsible for symptoms like nausea and emesis. Systematic studies of the in vivo effects of the amaryllidaceaeous-type alkaloids are not available. Therefore, in an open, prospective, randomized and controlled trial we studied the dose-effect relationship of lycorine-induced nausea and emesis and the toxicokinetics of lycorine in beagle dogs. Subcutaneously administered lycorine-induced nausea and emesis starting at 0.5 mg/kg body weight reaching statistical significance at 1.0 mg/kg. The maximum emetic dose of lycorine (ED(100)) was 2 mg/kg body weight. There was a correlation between dose and nausea score as well as between dose and number of the induced emetic events. Nausea and emesis were short-lasting and occurred not later than 2.5 h post dose. Lycorine showed linear plasma kinetics with a mean elimination half-life of 0.67 and 0.3 h after single s.c. and i.v. administration, compatible with the clinical course of nausea and emesis. The mean oral bioavailability was calculated to be about 40%. Biochemical and haematological parameters of safety showed no pathological signs. The results provide evidence that lycorine can be considered as a main, if not the crucial constituent responsible for nausea and emesis in human and animals in poisoning due to ingestion of plant material of the Amaryllidaceae.

Segment-dependent Expression of Muscarinic Acetylcholine Receptors and G-protein Coupling in the Equine Respiratory Tract

Muscarinic receptors are considered to be of comparable clinical importance in chronic obstructive pulmonary disease (COPD) in equines and in humans. At present, data are scarce on the expression and distribution of probable subtypes of these receptors and their signalling pathways in airway segments, including lung parenchyma and bronchial and tracheal epithelium with the underlying smooth muscle in horses. Specific [N-methyl-3H]scopolamine chloride ([3H]NMS) binding to all three tissues was saturable and of high affinity, with KD values ranging between 1.6±0.7 and 1.9±0.3 nmol/L. [3H]NMS binding identified a higher density of total muscarinic receptors (fmol/mg protein) in the trachea (720±59 nmol/L) than in bronchi (438±48 nmol/L) or lung (22 ± 3 nmol/L). Competitive binding studies using [3H]NMS and the unlabelled subtype-selective antagonists pirenzepine and telenzepine (M1), methoctramine and himbacine (M2), 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) (M3), tropicamide (M4) and mamba toxin (MT-3) (M4) indicated the presence of at least three muscarinic receptor subtypes in peripheral lung tissue (50:40:24–28%: M2>M3>M1), whereas in bronchus and trachea M2 subtypes (87–90%) predominated over M3 (14–22%), and M1 subtypes were lacking. No differences were found between tissues in high-affinity binding sites for carbachol in the absence (31–36%) or presence of guanosine 5′-triphosphate (GTP) (∼100%). Western blotting for G-protein α-subunits showed a much more robust expression of Gαi1/2 in the trachea (with highest receptor density) than in the lung or bronchi, whereas Gαs-protein was dominantly expressed in bronchus. Concomitantly, carbachol inhibited isoproterenol- and GTP-stimulated adenylyl cyclase activity with increasing muscarinic receptor expression (trachea > bronchi > lung). We conclude that the expression and signalling pathways of muscarinic receptors in the equine respiratory tract are segment-dependent. These receptors might contribute to the pathogenesis of COPD in the horse and could provide potential drug targets for the therapeutic use of anticholinergics in this species.

Serum thyroid hormone, insulin, glucose, triglycerides and protein concentrations in normal horses: Association with topical dexamethasone usage

The aim of this study was to determine if topical application of dexamethasone affected the serum concentrations of thyroid hormones (triiodothyronine T(3) and thyroxine T(4)), glucose, triglycerides, total protein and insulin in normal horses. Ten horses were treated twice daily for 10 days with 50 g dexamethasone using an ointment formulation. Thyroid hormones and insulin were assayed using standard radioimmunoassay methods, while glucose, triglycerides and total protein were determined using a standard enzymatic method and the Biuret reaction, respectively. An increase in serum glucose and triglyceride concentrations was accompanied by 2-6-fold increases in serum insulin concentrations, but there was no change in serum total protein concentration. Insulin secretion increased with concomitant hyperglycemia and hypertriglyceridemia. A non-significant decline in T(4) secretion was noted. Serum T(3) and T(4) concentrations declined continuously below baseline values from 48 h. Glucose and insulin levels returned to baseline values 3 days after treatment withdrawal, whereas triglycerides reverted to baseline by 7 days. In contrast, baseline values of serum T(3) and T(4) were not reached by 20 days following drug withdrawal. The results indicated that topical administration of dexamethasone affected thyroid function and physiological metabolic functions, which may have implications for potential doping cases in racing horses.

Influence of topical dexamethasone applications on insulin, glucose, thyroid hormone and cortisol levels in dogs

The influence of two topical dexamethasone applications (dermal and ototopical) on plasma insulin, glucose, thyroid hormone and cortisol levels was investigated in beagle dogs. Both treatments significantly decreased basal cortisol values, associated with exaggerated rise in insulin (approximately 50%), together with unchanged serum glucose levels. Dermal dexamethasone quickly decreased plasma thyroxin (T4) levels; whereas dexamethasone in ear drops gradually inhibited time-dependently T4 release (18-50%). Both formulations blunted plasma triiodothyronine (T3) levels but the response induced by dermal dexamethasone was stronger than by dexamethasone ear drops. Upon drug withdrawal, insulin secretion returned to baseline a week after treatment cessation, while cortisol, T4 and T3 levels did not reach baseline values. These results suggest that topical glucocorticoids unexpectedly trigger secondary hypothyroidism with concomitant suppression of hypothalamic-pituitary-adrenal axis but sensitize the endocrine pancreas, thus, their application needs careful evaluation for surprisingly different effects on endocrine stress axis activity.

Dexamethasone-Induced Increase in Lymphocyte β-Adrenergic Receptor Density and cAMP Formation in vivo

The influence of dexamethasone on the density of β2-adrenergic receptors (β2-adrenergic receptors (β2AR) and on the intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) response was studied in equine lymphocytes in vivo. Dexamethasone (0.1 mg/kg/day, 1–5 days) raised the number of β2AR – Bmax as assessed by (–)-[125I]iodocyanopindolol binding (ICYP) – to 2.5- to 3.5-fold as compared with control values. The increase in β2AR number was fast (342 ± 49 vs. 960 ± 103 binding sites/lymphocyte after 24 h), reaching a maximum between 48 and 96 h (342 ± 49 vs. 1,289 ± 150 and 1,106 ± 68 binding sites/lymphocyte, respectively). The isoprenaline-induced cAMP accumulation (measured by a [3H]-cAMP radioimmunoassay system) was concomitantly enhanced by dexamethasone (1.5- to 2.4-fold). Both parameters were reversible to a similar rate at dexamethasone withdrawal. The changes in the functional responsiveness of lymphocytes were not reflected by changes in the binding affinity for ICYP of β2AR. These results demonstrate the in vivo glucocorticoid-mediated regulation of β2AR in equine lymphocytes which has already been suggested on the basis of in vitro observations in other tissues.