Getu Abraham

The presumed atypical chemokine receptor CXCR7 signals through Gi/o proteins in primary rodent astrocytes and human glioma cells

SDF‐1/CXCL12 binds to the chemokine receptors, CXCR4 and CXCR7, and controls cell proliferation and migration during development, tumorigenesis, and inflammatory processes. It is currently assumed that CXCR7 would represent an atypical or scavenger chemokine receptor which modulates the function of CXCR4. Contrasting this view, we demonstrated recently that CXCR7 actively mediates SDF‐1 signaling in primary astrocytes. Here, we provide evidence that CXCR7 affects astrocytic cell signaling and function through pertussis toxin‐sensitive Gi/o proteins. SDF‐1‐dependent activation of Gi/o proteins and subsequent increases in intracellular Ca2+ concentration persisted in primary rodent astrocytes with depleted expression of CXCR4, but were abolished in astrocytes with depleted expression of CXCR7. Moreover, CXCR7‐mediated effects of SDF‐1 on Erk and Akt signaling as well as on astrocytic proliferation and migration were all sensitive to pertussis toxin. Likewise, pertussis toxin abolished SDF‐1‐induced activation of Erk and Akt in CXCR7‐only expressing human glioma cell lines. Finally, consistent with a ligand‐biased function of CXCR7 in astrocytes, the alternate CXCR7 ligand, I‐TAC/CXCL11, activated Erk and Akt through β‐arrestin. The demonstration that SDF‐1‐bound CXCR7 activates Gi/o proteins in astrocytes could help to explain some discrepancies previously observed for the function of CXCR4 and CXCR7 in other cell types.

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.

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.

Effects of autologous bone marrow stem cell transplantation on beta-adrenoceptor density and electrical activation pattern in a rabbit model of non-ischemic heart failure

BackgroundSince only little is known on stem cell therapy in non-ischemic heart failure we wanted to know whether a long-term improvement of cardiac function in non-ischemic heart failure can be achieved by stem cell transplantation.MethodsWhite male New Zealand rabbits were treated with doxorubicine (3 mg/kg/week; 6 weeks) to induce dilative non-ischemic cardiomyopathy. Thereafter, we obtained autologous bone marrow stem cells (BMSC) and injected 1.5–2.0 Mio cells in 1 ml medium by infiltrating the myocardium via a left anterolateral thoracotomy in comparison to sham-operated rabbits. 4 weeks later intracardiac contractility was determined in-vivo using a Millar catheter. Thereafter, the heart was excised and processed for radioligand binding assays to detect β1- and β2-adrenoceptor density. In addition, catecholamine plasma levels were determined via HPLC. In a subgroup we investigated cardiac electrophysiology by use of 256 channel mapping.ResultsIn doxorubicine-treated animals β-adrenoceptor density was significantly down-regulated in left ventricle and septum, but not in right ventricle, thereby indicating a typical left ventricular heart failure. Sham-operated rabbits exhibited the same down-regulation. In contrast, BMSC transplantation led to significantly less β-adrenoceptor down-regulation in septum and left ventricle. Cardiac contractility was significantly decreased in heart failure and sham-operated rabbits, but was significantly higher in BMSC-transplanted hearts. Norepinephrine and epinephrine plasma levels were enhanced in heart failure and sham-operated animals, while these were not different from normal in BMSC-transplanted animals. Electrophysiological mapping revealed unaltered electrophysiology and did not show signs of arrhythmogeneity.ConclusionBMSC transplantation improves sympathoadrenal dysregualtion in non-ischemic heart failure.

Regulation of equine lymphocyte β-adrenoceptors under the influence of clenbuterol and dexamethasone

In 12 healthy horses, the effects of the beta2-agonist clenbuterol and the glucocorticoid dexamethasone on the lymphocyte beta2-adrenoceptor density and affinity (determined by (-)-[125I]-iodocyanopindolol binding) as well as its responsiveness (assessed by lymphocyte cyclic AMP [cAMP] responses to 10 micromol/l (-)-isoprenaline) were studied. Clenbuterol treatment, 2 x 0.8 microg/kg/day i.v. for 12 days, decreased significantly ICYP binding sites by approximately 30-40%; concomitantly, lymphocyte cAMP response to (-)-isoprenaline was reduced. After withdrawal of clenbuterol, beta2-adrenoceptor density and responsiveness gradually increased, reaching predrug levels after 4 days. The effects of dexamethasone on clenbuterol-induced desensitisation were further investigated. Administration of dexamethasone (1 x 0.1 mg/kg/day, i.v. for 5 days) immediately after clenbuterol withdrawal accelerated beta2-adrenoceptor recovery: only 24 h after administration dexamethasone restored the number of binding sites and cAMP response to (-)-isoprenaline to levels statistically indistinguishable from values before clenbuterol treatment. Three days after dexamethasone administration, lymphocyte beta2-adrenoceptors were further increased about 2-fold the pretreatment values, and this increase declined gradually after dexamethasone withdrawal, reaching baseline values after 4 days. Furthermore, in groups exposed simultaneously to both drugs, dexamethasone completely prevented clenbuterol-induced decrease in lymphocyte beta2-adrenergic receptor density and responsiveness. No significant change was observed in the dissociation constant for ICYP in any of the situations. We conclude that dexamethasone (glucocorticoids) can reverse and prevent Clenbuterol-induced desensitisation (down-regulation) of the lymphocyte beta2-adrenoceptors and therefore, a combined therapy with clenbuterol and dexamethasone may be potentially beneficial in horses suffering from chronic obstructive pulmonary disease (COPD).

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.

Cardiac β-adrenoceptor changes in monocrotaline-treated rats: Differences between membrane preparations from whole ventricles and isolated ventricular cardiomyocytes

In monocrotaline (MCT)-treated rats the &bgr;-adrenoceptor-G-protein-adenylyl cyclase system—determined in crude membrane preparations from whole ventricular tissue—was desensitized not only in right (RV) but also in left ventricles (LV). This study aimed to assess the specific contribution of cardiomyocytes to these &bgr;-adrenoceptor changes. Six-week-old male Wistar rats were treated with 60 mg/kg body weight MCT intraperitoneally; within 4–6 weeks, rats developed marked RV hypertrophy. Cardiomyocytes were isolated from RVs and LVs. In RV cardiomyocytes of MCT-treated rats, &bgr;-adrenoceptor density was significantly reduced whereas it was unaltered in LV cardiomyocytes. Reduction of RV cardiomyocyte &bgr;-adrenoceptors was due to a selective &bgr;1-adrenoceptor reduction. Isoprenaline (100 &mgr;M)–induced cAMP increase was significantly reduced in RV but not in LV cardiomyocytes of MCT-treated rats. G protein–coupled receptor kinase activity was increased in RV but not in LV cardiomyocytes. &agr;1-Adrenoceptor density and noradrenaline-induced increase in inositol phosphate formation were significantly reduced only in RV but not in LV cardiomyocytes from MCT-treated rats. It is concluded that in cardiomyocytes of MCT-treated rats, cardiac &bgr;-adrenoceptors and &agr;1-adrenoceptors are chamber-specifically desensitized only in the RV. Thus, changes in cardiac &bgr;-adrenoceptors determined in membrane preparations from whole tissue homogenates do not correctly reflect changes occurring in cardiomyocytes.

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.

Comparative study of the effects of fetal bovine serum versus horse serum on growth and differentiation of primary equine bronchial fibroblasts

BackgroundAirway fibroblasts have become a critical addition to all facets of structural lung tissue changes such as in human asthma and chronic obstructive pulmonary disease, but little is known about their role in the equine recurrent airway obstruction, a disease that resembles to the human asthma. Since the equine bronchial fibroblasts (EBF) have not been isolated and characterized yet, the use of defined medium was investigated.ResultsPrimary EBF were cultured on non-collagen coated flasks without serum or in the presence of fetal bovine serum (FBS) or horse serum (HS) or in serum depleted medium. EBF cultured in serum-free basal media and those serum deprived were not able to proliferate and even exhibited considerable cell death. In media containing FBS or HS, proliferation of the cells was reproducible between different primary cultures and cells demonstrated expression of vimentin. Large variations were found in the ability of FBS and HS to support growth and differentiation of EBF in monolayer culture. Indications of growth-promoting actions, increasing passage number as well as maintaining fibroblast morphology were found rather in FBS than in HS. EBF culturing in HS needed longer doubling and confluence time. The protein content of the cell pellets was higher in EBF cultured in medium containing HS than FBS. Alpha-smooth muscle actin seemed to be less expressed in EBF cultured in medium containing FBS than those in HS.ConclusionsIn sum, serum addition to basal EBF medium enhanced EBF differentiation into myofibroblasts, and these findings are useful to develop in vitro fibroblast culture models that mimic in vivo physiological processes and to study airway disease mechanisms and remodeling.