Functional cross-talk between cytokine receptors revealed by activating mutations in the extracellular domain of the beta-subunit of the GM-CSF receptor

Several reports have suggested an interaction between the erythropoietin receptor (EpoR) and the shared signaling subunit (hbeta(c)) of the human granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 receptors, although the functional consequences of this interaction are unclear. We previously showed that in vivo expression of constitutively active extracellular (EC) mutants of hbeta(c) induces erythrocytosis and Epo independence of erythroid colony-forming units (CFU-E). This occurs despite an apparent requirement of these mutants for the GM-CSF receptor alpha-subunit (GMRalpha), which is not expressed in CFU-E. Here, we show that coexpression of hbeta(c) EC mutants and EpoR in BaF-B03 cells, which lack GMRalpha, results in factor-independent proliferation and JAK2 activation. Mutant receptors that cannot activate JAK2 fail to produce a functional interaction. As there is no detectable phosphorylation of hbeta(c). on intracellular tyrosine residues, EpoR displays constitutive tyrosine phosphorylation. These observations suggest that JAK2 activation mediates cross-talk between EC mutants of hbeta(c) and EpoR. The implications of these data are discussed as are our findings that activated hbeta(c) mutants can functionally interact with certain other cytokine receptors.

Ca2+-activated K+ channels in rat otic ganglion cells: Role of Ca2+ entry via Ca2+ channels and nicotinic receptors

1. Intracellular recordings were made from neurones in the rat otic ganglion in vitro in order to investigate their morphological, physiological and synaptic properties. We took advantage of the simple structure of these cells to test for a possible role of calcium influx via nicotinic acetylcholine receptors during synaptic transmission. 2. Cells filled with biocytin comprised a homogeneous population with ovoid somata and sparse dendritic trees. Neurones had resting membrane potentials of -53 +/- 0.7 mV (n = 69), input resistances of 112 + 7 M Omega, and membrane time constants of 14 +/- 0.9 ms (n = 60). Upon depolarization, all cells fired overshooting action potentials which mere followed by an apamin-sensitive after-hyperpolarization (AHP). In response to a prolonged current injection, all neurones fired tonically. 3. The repolarization phase of action potentials had a calcium component which was mediated by N-type calcium channels. Application of omega-conotoxin abolished both the repolarizing hump and the after-hgrperpolarization suggesting that calcium influx via N-type channels activates SK-type calcium-activated potassium channels which underlie the AHP. 4. The majority (70%) of neurones received innervation from a single preganglionic fibre which generated a suprathreshold excitatory postsynaptic potential mediated by nicotinic acetylcholine receptors. The other 30% of neurones also had one or more subthreshold nicotinic inputs. 5. Calcium influx via synaptic nicotinic receptors contributed to the AHP current, indicating that this calcium has access to the calcium-activated potassium channels and therefore plays a role in regulating cell excitability.

Human leukemia inhibitory factor upregulates LDL receptors on liver cells and decreases serum cholesterol in the cholesterol-fed rabbit

In a previous study, we found that the cytokine (human) leukemia inhibitory factor (hLIF) significantly reduced plasma cholesterol levels and the accumulation of lipid in aortic tissues of cholesterol-fed rabbits after 4 weeks of treatment. The mechanisms by which this occurs were investigated in the present study. This involved examining the effect of hLIF on (1) the level of plasma cholesterol at different times throughout the 4-week treatment and diet period; (2) smooth muscle cell (SMC) and macrophage-derived foam cell formation in vitro; and (3) LDL receptor expression and uptake in the human hepatoma cell line HepG2. At time zero, an osmotic minipump (2-mL capacity; infusion rate, 2.5 mu L/h; 28 days) containing either hLIF (30 mu g.kg(-1).d(-1)) or saline was inserted into the peritoneal cavity of New Zealand White rabbits (N=24). Rabbits were divided into four groups of six animals each. Group 1 received a normal diet/saline; group 2, a normal diet/hLIF; group 3, a 1% cholesterol diet/saline; and group 4, a 1% cholesterol diet/hLIF. hLIF had no effect on the plasma lipids or artery wall of group 2 rabbits (normal diet). However, in group 4 rabbits, plasma cholesterol levels and the percent surface area of thoracic aorta covered by fatty streaks was decreased by approximate to 30% and 80%, respectively, throughout all stages of the 4-week treatment period. In vitro, hLIF failed to prevent lipoprotein uptake by either SMCs or macrophages (foam cell formation) when the cells were exposed to P-VLDL for 24 hours. In contrast, hLIF (100 ng/mL) added to cultured human hepatoma HepG2 cells induced a twofold or threefold increase in intracellular lipid accumulation in the medium containing 10% lipoprotein-deficient serum or 10% fetal calf serum, respectively. This was accompanied by a significant non-dose-dependent increase in LDL receptor expression in hLIF-treated HepG2 cells incubated with LDL (20 mu g/mL) when compared with controls (P

Expression of activated mutants of the human interleukin-3/interleukin-5 granulocyte-macrophage colony-stimulating factor receptor common beta subunit in primary hematopoietic cells induces factor-independent proliferation and differentiation

To date, several activating mutations have been discovered in the common signal-transducing subunit (h beta c) of the receptors for human granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5. Two of these, Fl Delta and 1374N, result in a 37 amino acid duplication and a single amino acid substitution in the extracellular domain of h beta c, respectively. A third, V449E, results in a single amino acid substitution in the transmembrane domain, Previous studies comparing the activity of these mutants in different hematopoietic cell lines imply that the transmembrane and extracellular mutations act by different mechanisms and suggest the requirement for cell type-specific molecules in signalling. To characterize the ability of these mutant hpc subunits to mediate growth and differentiation of primary cells and hence investigate their oncogenic potential, we have expressed all three mutants in primary murine hematopoietic cells using retroviral transduction. It is shown that, whereas expression of either extracellular hpc mutant confers factor-independent proliferation and differentiation on cells of the neutrophil and monocyte lineages only, expression of the transmembrane mutant does so on these lineages as well as the eosinophil, basophil, megakaryocyte, and erythroid lineages, Factor-independent myeloid precursors expressing the transmembrane mutant display extended proliferation in liquid culture and in some cases yielded immortalized cell lines. (C) 1997 by The American Society of Hematology.

The expression of fibroblast growth factors and their receptors in the embryonic and neonatal mouse inner ear

Four different fibroblast growth factor receptors (FGFR) are known, three of which have splice variants (known as the b and c variants) in the FGF-binding domain, to give different patterns of sensitivity to the different FGFs. The expression of the b and c variants of the FGF receptors. together with the expression of the ligands FGF1. FGF2, FGF3, FGF7, FGF8b and FGF8c, was determined by quantitative reverse transcription-polymerase chain reaction in developing whole mouse inner ears, and in dissected components of the postnatal mouse inner ear. At embryonic age (E)10.5 days, when the otocyst is a simple closed sac, the receptor most heavily expressed was FGFR2b, relative to the postnatal day 0 level. Over the period E10.5-E12.5. during which the structures of the inner ear start to form, the expression of the different FGF receptors increased 10(2)-10(4) fold per unit of tissue, and there was a gradual switch towards expression of the 'c' splice variants of FGFR2 and FGFR3 rather than the 'b' variants. At E10.5, the ligands most heavily expressed, relative to the postnatal day 0 level, were FGF3, FGF8b and FGF8c. In the postnatal inner eat. the patterns of expression of receptors and ligands tended to be correlated, such that receptor variants were expressed in the same regions as the ligands that are known to activate them effectively. The neural/sensory region expressed high levels of FGFR3c, and high levels of the ligand FGF8b. The same area also expressed high levels of FGFR1b and FGFR2b, and high levels of FGF3. The lateral wall of the cochlea (including the stria vascularis and the spiral ligament) expressed high levels of FGFR1c and FGF1. 11 is suggested that the different FGF receptors and ligands are expressed in a spatially coordinated pattern to selectively program cochlear development. (C) 2001 Elsevier Science B.V. All rights reserved.

Crown ether appended cyclam receptors for cationic guests

A series of crown ether appended macrocyclic amines has been prepared comprising benzo-12-crown-4, benzo-15-crown-5, or benzo-18-crown-6 attached to a diamino-substituted cyclam. The Co-III complexes of these three receptors have been prepared and characterized spectroscopically and structurally. Crystal structures of each receptor in complex with an alkali metal ion and structures of the benzo-12-crown-4 and benzo-15-crown-5-receptors without guest ions are reported. 2D NMR and molecular mechanics modeling have been used to examine conformational variations upon guest ion complexation. Addition of cations to these receptors results in an appreciable anodic shift in the Co-III:II 11 redox potential, even in aqueous solution, but little cation selectivity is observed. Evidence for complex formation has been corroborated by Na-23 and Li-7 NMR spectroscopy and electrospray mass spectrometry.

Morphine-3-glucuronide's neuro-excitatory effects are mediated via indirect activation of N-methyl-D-aspartic acid receptors: Mechanistic studies in embryonic cultured hippocampal neurones

Indirect evidence indicates that morphine-3-glucuronide (M3G) may contribute significantly to the neuro-excitatory side effects (myoclonus and allodynia) of large-dose systemic morphine. To gain insight into the mechanism underlying M3G' s excitatory behaviors, We used fluo-3 fluorescence digital imaging techniques to assess the acute effects of M3G (5-500 muM) on the cytosolic calcium concentration ([Ca2+](CYT)) in cultured embryonic hippocampal neurones. Acute (3 min) exposure of neurones to M3G evoked [Ca2+](CYT) transients that were typically either (a) transient oscillatory responses characterized by a rapid increase in [Ca2+](CYT) oscillation amplitude that was sustained for at least similar to30 s or (b) a sustained increase in [Ca2+](CYT) that slowly recovered to baseline. Naloxone-pretreatment decreased the proportion of M3G-responsive neurones by 10%-25%, implicating a predominantly non-opioidergic mechanism. Although the naloxone-insensitive M3G-induced increases in [Ca2+](CYT) were completely blocked by N-methyl-D-aspartic acid (NMDA) antagonists and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (alphaamino-3-hydroxy-5-methyl-4-isoxazolepropiordc acid/ kainate antagonist), CNQX did not block the large increase in [Ca2+](CYT) evoked by NMDA (as expected), confirming that N13G indirectly activates the NMDA receptor. Additionally, tetrodotoxin (Na+ channel blocker), baclofen (gamma-aminobutyric acid, agonist), MVIIC (P/Q-type calcium channel blocker), and nifedipine (L-type calcium channel blocker) all abolished M3G-induced increases in [Ca2+](CYT), suggesting that M3G may produce its neuro-excitatory effects by modulating neurotransmitter release. However, additional characterization is required.

Expression and function of neuronal nicotinic ACh receptors in rat microvascular endothelial cells

The expression and function of nicotinic ACh receptors (nAChRs) in rat coronary microvascular endothelial cells (CMECs) were examined using RT-PCR and whole cell patch-clamp recording methods. RT-PCR revealed expression of mRNA encoding for the subunits alpha(2), alpha(3), alpha(4), alpha(5), alpha(7), beta(2), and beta(4) but not beta(3). Focal application of ACh evoked an inward current in isolated CMECs voltage clamped at negative membrane potentials. The current-voltage relationship of the ACh-induced current exhibited marked inward rectification and a reversal potential (E-rev) close to 0 mV. The cholinergic agonists nicotine, epibatidine, and cytisine activated membrane currents similar to those evoked by ACh. The nicotine-induced current was abolished by the neuronal nAChR antagonist mecamylamine. The direction and magnitude of the shift in E-rev of nicotine-induced current as a function of extracellular Na+ concentration indicate that the nAChR channel is cation selective and follows that predicted by the Goldman-Hodgkin-Katz equation assuming K+/Na+ permeability ratio of 1.11. In fura-2-loaded CMECs, application of ACh, but not of nicotine, elicited a transient increase in intracellular free Ca2+ concentration. Taken together, these results demonstrate that neuronal nAChR activation by cholinergic agonists evokes an inward current in CMECs carried primarily by Na+, which may contribute to the plasma nicotine-induced changes in microvascular permeability and reactivity induced by elevations in plasma nicotine.

Comparative agonist/antagonist responses in mutant human C5a receptors define the ligand binding site

The C terminus is responsible for all of the agonist activity of C5a at human C5a receptors (C5aRs). In this report we have mapped the ligand binding site on the C5aR using a series of agonist and antagonist peptide mimics of the C terminus of C5a as well as receptors mutated at putative interaction sites ( Ile(116), Arg(175), Arg(206), Glu(199), Asp(282), and Val(286)). Agonist peptide 1 (Phe-Lys-Pro-D-cyclohexylalanine-cyclohexylalanine-D-Arg) can be converted to an antagonist by substituting the bulkier Trp for cyclohexylalanine at position 5 ( peptide 2). Conversely, mutation of C5aR transmembrane residue Ile(116) to the smaller Ala (I116A) makes the receptor respond to peptide 2 as an agonist (Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J., and Bourne, H. R. (2001) J. Biol. Chem. 276, 3394 - 3400). However, a potent cyclic hexapeptide antagonist, Phe-cyclo-[Orn-Pro-D-cyclohexylalanine-Trp-Arg] ( peptide 3), derived from peptide 2 and which binds to the same receptor site, remains a full antagonist at I116AC5aR. This suggests that although the residue at position 5 might bind near to Ile(116), the latter is not essential for either activation or antagonism. Arg(206) and Arg(175) both appear to interact with the C-terminal carboxylate of C5a agonist peptides, suggesting a dynamic binding mechanism that may be a part of a receptor activation switch. Asp(282) has been previously shown to interact with the side chain of the C-terminal Arg residue, and Glu(199) may also interact with this side chain in both C5a and peptide mimics. Using these interactions to orient NMR-derived ligand structures in the binding site of C5aR, a new model of the interaction between peptide antagonists and the C5aR is presented.

Pharmacological evaluation of an [I-123] labelled imidazopyridine-3-acetamide for the study of benzodiazepine receptors

In vitro binding of the iodinated imidazopyri dine, N',N'-dimethyl-6-methyl-(4'-[I-123]iodophenyl)imidazo[1,2-a]pyridine-3-acetamide [I-123]IZOL to benzodiazepine binding sites on brain cortex, adrenal and kidney membranes is reported. Saturation experiments showed that [I-123]IZOL, bound to a single class of binding site (n(H)=0.99) on adrenal and kidney mitochondrial membranes with a moderate affinity (K-d=30 nM). The density of binding sites was 22 +/- 6 and 1.2 +/- 0.4 pmol/mg protein on adrenal and kidney membranes, respectively. No specific binding was observed in mitochondrial-synaptosomal membranes of brain cortex. In biodistribution studies in rats, the highest uptake of [I-123]IZOL was found 30 min post injection in adrenals (7.5% ID/g), followed by heart, kidney, lung (1% ID/g) and brain (0.12% ID/g), consistent with the distribution of peripheral benzodiazepine binding sites. Pre-administration of unlabelled IZOL and the specific PBBS drugs, PK 11195 and Ro 5-4864 significantly reduced the uptake of [I-123]IZOL by 30% (p < 0.05) in olfactory bulbs and by 51-86% (p < 0.01) in kidney, lungs, heart and adrenals, while it increased by 30% to 50% (p < 0.01) in the rest of the brain and the blood. Diazepam, a mixed CBR-PBBS drug, inhibited the uptake in kidney, lungs, heart, adrenals and olfactory bulbs by 32% to 44% (p < 0.01) but with no effect on brain uptake and in blood concentration. Flumazenil, a central benzodiazepine drug and haloperidol (dopamine antagonist/sigma receptor drug) displayed no effect in [I-123]IZOL in peripheral organs and in the brain. [I-123]IZOL may deserve further development for imaging selectively peripheral benzodiazepine binding sites. (c) 2006 Elsevier Inc. All rights reserved.

Protease-activated receptor-2 peptides activate neurokinin-1 receptors in the mouse isolated trachea

Protective roles for protease-activated receptor-2 (PAR2) in the airways including activation of epithelial chloride (Cl-) secretion are based on the use of presumably PAR(2)-selective peptide agonists. To determine whether PAR(2) peptide-activated Cl- secretion from mouse tracheal epithelium is dependent on PAR(2), changes in ion conductance across the epithelium [short-circuit current (I-SC)] to PAR(2) peptides were measured in Ussing chambers under voltage clamp. In addition, epithelium and endothelium-dependent relaxations to these peptides were measured in two established PAR(2) bioassays, isolated ring segments of mouse trachea and rat thoracic aorta, respectively. Apical application of the PAR(2) peptide SLIGRL caused increases in I-SC, which were inhibited by three structurally different neurokinin receptor-1 (NK1R) antagonists and inhibitors of Cl- channels but not by capsaicin, the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37), or the nonselective cyclooxygenase inhibitor indomethacin. Only high concentrations of trypsin caused an increase in I-SC but did not affect the responses to SLIGRL. Relaxations to SLIGRL in the trachea and aorta were unaffected by the NK1R antagonist nolpitantium (SR 140333) but were abolished by trypsin desensitization. The rank order of potency for a range of peptides in the trachea I-SC assay was 2-furoyl-LIGRL > SLCGRL > SLIGRL > SLIGRT > LSIGRL compared with 2-furoyl-LIGRL > SLIGRL > SLIGRT > SLCGRL (LSIGRL inactive) in the aorta relaxation assay. In the mouse trachea, PAR(2) peptides activate both epithelial NK1R coupled to Cl- secretion and PAR(2) coupled to prostaglandin E-2-mediated smooth muscle relaxation. Such a potential lack of specificity of these commonly used peptides needs to be considered when roles for PAR(2) in airway function in health and disease are determined.

Synaptic activation of transient receptor potential channels by metabotropic glutamate receptors in the lateral amygdala

Classical mammalian transient receptor potential channels form non-selective cation channels that open in response to activation of phospholipase C-coupled metabotropic receptors, and are thought to play a key role in calcium homeostasis in non-excitable cells. Within the nervous system transient receptor potential channels are widely distributed but their physiological roles are not well understood. Here we show that in the rat lateral amygdala transient receptor potential channels mediate an excitatory synaptic response to glutamate. Activation of group l etabotropic glutamate receptors on pyramidal neurons in the lateral amygdala with either exogenous or synaptically released glutamate evokes an inward current at negative potentials with a current voltage relationship showing a region of negative slope and steep outward rectification. This current is blocked by inhibiting G protein function with GTP-beta-S, by inhibiting phospholipase C or by infusing transient receptor potential antibodies into lateral amygdala pyramidal neurons. Using RT-PCR and Western blotting we show that transient receptor potential 1, transient receptor potential 4 and transient receptor potential 5 are present in the lateral amygdala. Single cell PCR confirms the presence of transient receptor potential 1 and transient receptor potential 5 in pyramidal neurons and we show by co-immunoprecipitation that transient receptor potential 1 and transient receptor potential 5 co-assemble as a heteromultimers in the amygdala. These results show that in lateral amygdala pyramidal neurons synaptically released glutamate activates transient receptor potential channels, which we propose are likely to be heteromultimeric channels containing transient receptor potential 1 and transient receptor potential 5/transient receptor potential 4. (c) 2005 Published by Elsevier Ltd on behalf of IBRO.

Correlation of habitual physical activity levels with flow mediated dilation of the brachial artery in 5-10 year old children

Endothelial dysfunction is an early key event of atherogenesis. Both fitness level and exercise intervention have been shown to positively influence endothelial function. In a cross-sectional study of 47 children, the relationship between habitual physical activity and flow-mediated dilation (FMD) of the brachial artery was explored. Habitual physical activity levels (PALs) were assessed using a validated stable isotope technique, and FMD of the brachial artery was measured via high-resolution ultrasound. The results showed that habitual physical activity significantly correlated with FMD (r=0.39, P=0.007), and remained the most influential variable on dilation in multivariate analysis. Although both fitness level and exercise intervention have previously been shown to positively influence FMD, this is the first time that a relationship with normal PALs has been investigated, especially, at such a young age. These data support the concept that physical activity exerts its protective effect on cardiovascular health via the endothelium and add further emphasis to the importance of physical activity in childhood.

Habitual physical activity and physical activity intensity: their relation to body composition in 5.0-10.5 year old children

Background: Concerns of a decrease in physical activity levels (PALs) of children and a concurrent increase in childhood obesity exist worldwide. The exact relation between these two parameters however has as yet to be fully defined in children. Objective: This study examined the relation in 47 children, aged 5–10.5 y (mean age 8.4plusminus0.9 y) between habitual physical activity, minutes spent in moderate, vigorous and hard intensity activity and body composition parameters. Design: Total energy expenditure (TEE) was calculated using the doubly labelled water technique and basal metabolic rate (BMR) was predicted from Schofield's equations. PAL was determined by PAL=TEE/BMR. Time spent in moderate, vigorous and hard intensity activity was determined by accelerometry, using the Tritrac-R3D. Body fatness and body mass index (BMI) were used as the two measures of body composition. Results: Body fat and BMI were significantly inversely correlated with PAL (r=-0.43, P=0.002 and r=-0.45, P=0.001). Times spent in vigorous activity and hard activity were significantly correlated to percentage body fat (r=-0.44, P=0.004 and r=-0.39, P=0.014), but not BMI. Children who were in the top tertiles for both vigorous activity and hard activity had significantly lower body fat percentages than those in the middle and lowest tertiles. Moderate intensity activity was not correlated with measures of body composition. Conclusions: As well as showing a significant relation between PAL and body composition, these data intimate that there may be a threshold of intensity of physical activity that is influential on body fatness. In light of world trends showing increasing childhood obesity, this study supports the need to further investigate the importance of physical activity for children.

Aldermaston Peace March April 5 1964 Brisbane Australia

Three men and a woman during Aldermaston Peace March, April 5 1964 in Brisbane Australia. The march covered the distance between Ipswich and Brisbane, Australia. Marchers walked in relays covering approximately two miles each. Most relay sections were sponsored by one or more individual organisations. Convoy of Volkswagon Beetles and FB Holdens can be seen following them. Facades of Elphinstones store, Masonic Centre and St Andrews can be seen on the left with the People's Palace in the distance and the Central Railway Station on the right.