Carbachol triggers RyR-dependent Ca(2+) release via activation of IP(3) receptors in isolated rat gastric myocytes.

Possible interactions between different intracellular Ca(2+) release channels were studied in isolated rat gastric myocytes using agonist-evoked Ca(2+) signals. Spontaneous, local Ca(2+) transients were observed in fluo-4-loaded cells with linescan confocal imaging. These were blocked by ryanodine (100 microM) but not by the inositol 1,4,5-trisphosphate receptor (IP(3)R) blocker, 2-aminoethoxydiphenyl borate (100 microM), identifying them as Ca(2+) sparks. Caffeine (10 mM) and carbachol (10 microM) initiated Ca(2+) release at sites which co-localized with each other and with any Ca(2+) spark sites. In fura-2-loaded cells extracellular 2-aminoethoxydiphenyl borate and intracellular heparin (5 mg ml(-1)) both inhibited the global cytoplasmic [Ca(2+)] transient evoked by carbachol, confirming that it was IP(3)R-dependent. 2-Aminoethoxydiphenyl borate and heparin also increased the response to caffeine. This probably reflected an increased Ca(2+) store content since 2-aminoethoxydiphenyl borate more than doubled the amplitude of transients evoked by ionomycin. Ryanodine completely abolished carbachol and caffeine responses but only reduced ionomycin transients by 30 %, suggesting that blockade of carbachol transients by ryanodine was not simply due to store depletion. Double labelling of IP(3)Rs and RyRs demonstrated extensive overlap in their distribution. These results suggest that carbachol stimulates Ca(2+) release through co-operation between IP(3)Rs and RyRs, and implicate IP(3)Rs in the regulation of Ca(2+) store content.

Cytokine gene polymorphisms in ischaemic heart disease:Investigation using family based tests of association

Atherosclerosis has an inflammatory basis, with cytokines, cellular adhesion molecules and pro-inflammatory cells having important roles in the initiation and progression of this process. Interleukin (IL) 6, IL-10 and transforming growth factor (TGF) β have been proposed as important modulators of the atherosclerotic process, with IL-6 having a pro-inflammatory, atherogenic effect and IL-10 and TGF-β having anti-inflammatory, protective roles. The possible role of functional polymorphisms in the promoter regions of the IL-6, IL-10 and TGF-β genes in the susceptibility to ischaemic heart disease (IHD) was investigated in a well-defined Irish population using two recently described family-based tests of association. We genotyped 1,012 individuals from 386 families with at least one member prematurely affected with IHD. Using the combined transmission disequilibrium test (TDT)/sib-TDT and the pedigree disequilibrium test, no association between any of the IL-6 -174G/C, IL-10 -1082G/A and TGF-β -509C/T polymorphisms and IHD was found. Our data demonstrate that, in an Irish population, these polymorphisms are not associated with IHD. © Springer-Verlag 2004.

Simultaneously multiply-configurable or superposed molecular logic systems composed of ICT (internal charge transfer) chromophores and fluorophores integrated with one- or two-ion receptors

Integrated "ICT chromophore-receptor" systems show ion-induced shifts in their electronic absorption spectra. The wavelength of observation can be used to reversibly configure the system to any of the four logic operations permissible with a single input (YES, NOT, PASS 1, PASS 0), under conditions of ion input and transmittance output. We demonstrate these with dyes integrated into Tsien's calcium receptor, 1-2. Applying multiple ion inputs to 1-2 also allows us to perform two- or three-input OR or NOR operations. The weak fluorescence output of 1 also shows YES or NOT logic depending on how it is configured by excitation and emission wavelengths. Integrated "receptor(1)-ICT chromophore-receptor(2)" systems 3-5 selectively target two ions into the receptor terminals. The ion-induced transmittance output of 3-5 can also be configured via wavelength to illustrate several logic types including, most importantly, XOR. The opposite effects of the two ions on the energy of the chromophore excited state is responsible for this behaviour. INHIBIT and REVERSE IMPLICATION are two of the other logic types seen here. Integration of XOR logic with a preceding OR operation can be arranged by using three ion inputs. The fluorescence output of these systems can be configured via wavelength to display INHIBIT or NOR logic under two-input conditions. The superposition or multiplicity of logic gate configurations is an unusual consequence of the ability to simultaneously observe multiple wavelengths.

An Immunohistochemical Study of the Distribution of the Measles Virus Receptors, CD46 and SLAM, In Normal Human Tissues and Subacute Sclerosing Panencephalitis

We have compared the expression of the known measles virus (MV) receptors, membrane cofactor protein (CD46) and the signaling lymphocyte-activation molecule (SLAM), using immunohistochemistry, in a range of normal peripheral tissues (known to be infected by MV) as well as in normal and subacute sclerosing panencephalitis (SSPE) brain. To increase our understanding of how these receptors could be utilized by wild-type or vaccine strains in vivo, the results have been considered with regard to the known route of infection and systemic spread of MV. Strong staining for CD46 was observed in endothelial cells lining blood vessels and in epithelial cells and tissue macrophages in a wide range of peripheral tissues, as well as in Langerhans' and squamous cells in the skin. In lymphoid tissues and blood, subsets of cells were positive for SLAM, in comparison to CD46, which stained all nucleated cell types. Strong CD46 staining was observed on cerebral endothelium throughout the brain and also on ependymal cells lining the ventricles and choroid plexus. Comparatively weaker CD46 staining was observed on subsets of neurons and oligodendrocytes. In SSPE brain sections, the areas distant from lesion sites and negative for MV by immunocytochemistry showed the same distribution for CD46 as in normal brain. However, cells in lesions, positive for MV, were negative for CD46. Normal brain showed no staining for SLAM, and in SSPE brain only subsets of leukocytes in inflammatory infiltrates were positive. None of the cell types most commonly infected by MV show detectable expression of SLAM, whereas CD46 is much more widely expressed and could fulfill a receptor function for some wild-type strains. In the case of wild-type stains, which are unable to use CD46, a further as yet unknown receptor(s) would be necessary to fully explain the pathology of MV infection.

Expression of vascular endothelial growth factor (VEGF) and its receptors is regulated in eyes with intra-ocular tumours

The immunolocalization and gene expression of vascular endothelial growth factor (VEGF) and its cognate tyrosine kinase receptors, Flt-1 and KDR, has been studied in ocular melanomas and retinoblastomas using in situ hybridization and immunohistochemistry. Tumour-related alterations in VEGF/VEGF-receptor expression have also been examined in separate and uninvolved iris, retina and choroid of the same eyes. Although VEGF immunoreactivity in the normal retina was virtually absent, low-level VEGF expression was evident in the ganglion cell-bodies, Müller cells and in a distinct population of amacrine cells. VEGF gene expression was absent in the iris and choroid of normal eyes. In tumour-bearing eyes, high levels of VEGF protein and gene expression were observed within the vascularized regions of the tumours, while the adjacent retina and choroid showed increased VEGF levels when compared with normals. Flt-1 and KDR gene expression and immunolocalization occurred in VEGF-expressing ganglion, Müller and amacrine cells in normal eyes. Within the intra-ocular tumours, VEGF-receptor gene expression and protein was evident in the endothelial cells and also in cells close to the vessels, while in the adjacent retina, Flt-1 and KDR levels were elevated over normal, especially in the blood vessels. Flt-1 and KDR were both observed at elevated levels in the choroid and iris blood vessels. This study suggests that VEGF, Flt-1 and KDR are expressed by neural, glial and vascular elements within normal human retina. Intra-ocular tumours demonstrate a high level of VEGF and VEGF-receptor expression; within uninvolved, spatially separate retina, choroid and iris in the same eyes, expression is also elevated, especially within the vasculature. Retinal vascular endothelia may respond to high intra-ocular levels of VEGF by increasing expression of their VEGF receptors, a phenomenon which could have relevance to neoplasm-related ocular neovascularization.

Cybr, a cytokine-inducible protein that binds cytohesin-1 and regulates its activity

Cytokines regulate lymphocyte development and differentiation, but precisely how they control these processes is still poorly understood. By using microarray technology to detect cytokine-induced genes, we identified a cDNA encoding Cybr, which was increased markedly in cells incubated with IL-2 and IL-12. The mRNA was most abundant in hematopoietic cells and tissues. The predicted amino acid sequence is similar to that of GRP-1-associated protein (GRASP), a recently identified retinoic acid-induced cytohesin-binding protein. Physical interaction, dependent on the coiled-coil domains of Cybr and cytohesin-1, was demonstrated by coimmunoprecipitation of the overexpressed proteins from 293T cells. Cytohesin-1, in addition to its role in cell adhesion, is a guanine nucleotide-exchange protein activator of ARF GTPases. Acceleration of guanosine 5'-O-(thiotriphosphate) binding to ARF by cytohesin-1 in vitro was enhanced by Cybr. Because the binding protein modified activation of ADP ribosylation factor by cytohesin-1, we designate this cytokine-inducible protein Cybr (cytohesin binder and regulator).

The chemoattractants, IL-8 and formyl-methionyl-leucyl-phenylalanine, regulate granulocyte colony-stimulating factor signaling by inducing suppressor of cytokine signaling-1 expression

Suppressors of cytokine signaling (SOCS) are encoded by immediate early genes known to inhibit cytokine responses in a classical feedback loop. SOCS gene expression has been shown to be induced by many cytokines, growth factors, and innate immune stimuli, such as LPS. In this paper, we report that the chemoattractants, IL-8 and fMLP, up-regulate SOCS1 mRNA in human myeloid cells, primary human neutrophils, PBMCs, and dendritic cells. fMLP rapidly up-regulates SOCS1, whereas the induction of SOCS1 upon IL-8 treatment is delayed. IL-8 and fMLP did not signal via Jak/STATs in primary human macrophages, thus implicating the induction of SOCS by other intracellular pathways. As chemoattractant-induced SOCS1 expression in neutrophils may play an important role in regulating the subsequent response to growth promoting cytokines like G-CSF, we investigated the effect of chemoattractant-induced SOCS1 on cytokine signal transduction. We show that pretreatment of primary human neutrophils with fMLP or IL-8 blocks G-CSF-mediated STAT3 activation. This study provides evidence for cross-talk between chemoattractant and cytokine signal transduction pathways involving SOCS proteins, suggesting that these chemotactic factors may desensitize neutrophils to G-CSF via rapid induction of SOCS1 expression.

Use of A192621 to provide evidence for involvement of endothelin ET(B)-receptors in endothelin-1-mediated cardiomyocyte hypertrophy.

Increased plasma levels of endothelin-1 correlate with the severity of left ventricular hypertrophy in vivo. The aim of the study was to determine the relative contribution of stimulation of endothelin ETA and endothelin ETB receptors, and the associated activation of protein kinase C, to the hypertrophic response initiated by endothelin-1 in adult rat ventricular cardiomyocytes maintained in culture (24 h). Endothelin-1 (10-7 M) increased the total mass of protein and the incorporation of [14C] phenylalanine into protein to 26% and 25% greater (P

Modulation of contractile function through NPY receptors during development of cardiomyocyte hypertrophy.

Severity of left ventricular hypertrophy (LVH) correlates with elevated plasma levels of neuropeptide Y (NPY) in hypertension. NPY elicits positive and negative contractile effects in cardiomyocytes through Y(1) and Y(2) receptors, respectively. This study tested the hypothesis that NPY receptor-mediated contraction is altered during progression of LVH. Ventricular cardiomyocytes were isolated from spontaneously hypertensive rats (SHRs) pre-LVH (12 weeks), during development (16 weeks), and at established LVH (20 weeks) and age-matched normotensive Wistar Kyoto (WKY) rats. Electrically stimulated (60 V, 0.5 Hz) cell shortening was measured using edge detection and receptor expression determined at mRNA and protein level. The NPY and Y(1) receptor-selective agonist, Leu(31)Pro(34)NPY, stimulated increases in contractile amplitude, which were abolished by the Y(1) receptor-selective antagonist, BIBP3226 [R-N(2)-(diphenyl-acetyl)-N-(4-hydroxyphenyl)methyl-argininamide)], confirming Y(1) receptor involvement. Potencies of both agonists were enhanced in SHR cardiomyocytes at 20 weeks (2300- and 380-fold versus controls). Maximal responses were not attenuated. BIBP3226 unmasked a negative contraction effect of NPY, elicited over the concentration range (10(-12) to 3 x 10(-9) M) in which NPY and PYY(3-36) attenuated the positive contraction effects of isoproterenol, the potencies of which were increased in cardiomyocytes from SHRs at 20 weeks (175- and 145-fold versus controls); maximal responses were not altered. Expression of NPY-Y(1) and NPY-Y(2) receptor mRNAs was decreased (55 and 69%) in left ventricular cardiomyocytes from 20-week-old SHRs versus age-matched WKY rats; parallel decreases (32 and 80%) were observed at protein level. Enhancement of NPY potency, producing (opposing) contractile effects on cardiomyocytes together with unchanged maximal response despite reduced receptor number, enables NPY to contribute to regulating cardiac performance during compensatory LVH.

DUB-3, a Cytokine inducible Deubiquitinating Enzyme That Blocks Profileration

Previous studies have identified the DUB family of cytokine-regulated murine deubiquitinating enzymes, which play a role in the control of cell proliferation and survival. Through data base analyses and cloning, we have identified a human cDNA (DUB-3) that shows significant homology to the known murine DUB family members. Northern blotting has shown expression of this gene in a number of tissues including brain, liver, and muscle, with two transcripts being apparent (1.6 and 1.7 kb). In addition, expression was observed in cell lines including those derived from a number of hematopoietic tumors such as the Burkitt's lymphoma cell line RAJI. We have also demonstrated that DUB-3, which was shown to be an active deubiquitinating enzyme, is induced in response to interleukin-4 and interleukin-6 stimulation. Finally, we have demonstrated that constitutive expression of DUB-3 blocks proliferation and can initiate apoptosis in both IL-3-dependent Ba/F3 cells and NIH3T3 fibroblasts. These findings suggest that human DUB-3, like the murine DUB family members, is transiently induced in response to cytokines and can, when constitutively expressed, block growth factor-dependent proliferation.