Intercellular signaling in neuronal-glial networks

Glial cells have recently been found to exhibit electrophysiological and metabolic responses to many neurotransmitters and neuromodulators. These findings have focused attention on the possibility that active signaling between neurons and glia could represent an important form of intercellular communication within the brain. Since glial and neuronal networks are both physically and metabolically interlinked, such intercellular signaling may represent a mechanism for inducing collective changes in the cellular physiology of neuronal and glial cell populations. Within the nervous tissue of both vertebrate and invertebrate organisms, glial cells are known to secrete extracellular signal molecules, modulate carbohydrate metabolism, and control the volume and ionic composition of extracellular space. In this paper, the roles that cytoplasmic [Ca2+] transients may play in regulating these glial cell functions are reviewed. Mechanisms by which intracellular Ca oscillations and intercellular Ca waves may be generated in neurotransmitter-stimulated glial cells are also discussed. In addition, it is proposed that rhythmic glial cell contractions and shape changes, which have been observed for many decades, are linked to Ca-induced secretion of ions, water, and neuroactive compounds. These activities represent mechanisms by which Ca-induced changes in glial cell physiology could potentially alter the excitability of neuronal networks.     

Interaction of invasive bacteria with host signaling pathways

PURPOSE: Programmed cell death (apoptosis) is the controlled death of cells that occurs with minimal collateral damage to surrounding cells or tissue during development, homeostasis, and wound healing. The authors hypothesize the keratocyte apoptosis is an initiating factor in the wound-healing response after refractive surgical procedures. To evaluate the effects of different corneal manipulations, keratocyte apoptosis was examined qualitatively and quantitatively after traditional epithelial scrape-photorefractive keratectomy (PRK), transepithelial PRK, removal of a cap of superficial cornea using a microkeratome, production of a flap of superficial cornea with a microkeratome, and laser-assisted in situ keratomileusis (LASIK) compared with unwounded controls in rabbit corneas. METHODS: Refractive surgical procedures or their components were performed in rabbit eyes. Keratocyte apoptosis was monitored using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling assay to detect DNA fragmentation. Cellular morphologic changes were evaluated by electron microscope examination. RESULTS: Keratocyte apoptosis was noted with each refractive procedure or corneal manipulation and was variable from eye to eye with each procedure. Transepithelial PRK was associated with the lowest levels of central corneal apoptosis, even if the stromal surface was scraped after the procedure. Keratocyte apoptosis is confined to the superficial stroma extending to a depth of approximately 50 microns to 75 microns after epithelial scrape-PRK and transepithelial PRK. Apoptosis was noted in the deeper central corneal keratocytes located anteriorly and posteriorly to the lamellar cut in LASIK. CONCLUSIONS: There are qualitative and quantitative differences in keratocyte apoptosis between LASIK, epithelial scrape-PRK, and transepithelial PRK. Epithelial injury is an important factor modulating keratocyte apoptosis. The level and distribution of keratocyte apoptosis, along with subsequent repopulation by activated stromal keratocytes, are likely to be important determinants of corneal wound healing associated with variability and regression after PRK and LASIK. Transepithelial PRK induces low levels of keratocyte apoptosis, and, therefore, this approach may be useful for treating higher levels of myopia and for retreatment after regression.     

Developmental regulation of cAMP signaling pathways in thymocyte development

Major developmental transitions in thymocyte differentiation are accompanied by sharp alterations in cAMP metabolism. We have analyzed the cAMP accumulation responses of cell populations representing successive stages of T-cell development, namely: immature TcR- thymocytes from SCID mice, proliferating cortical blasts, small cortical thymocytes, medullary thymocytes and peripheral T cells. We find that all classes of thymocytes exhibit higher cAMP synthesis in response to forskolin than peripheral T cells. In immature TcR- thymocytes, this high capacity is buffered by efficient phosphodiesterase activity, but in CD4+CD8+TcRlow thymocytes, phosphodiesterase activity becomes much less effective. Phosphodiesterase activity then rises again after positive selection. The ability of thymocytes to respond to prostaglandin E is regulated distinctly from their ability to respond to forskolin. Unlike forskolin, PGE1 induces cAMP synthesis to similar levels in all classes of thymocytes, possibly due to partial activation of phosphodiesterase in cortical thymocytes by PGE1. Finally, we report a novel effect of Ca2+/protein kinase C signaling on cAMP accumulation, which occurs selectively in the proliferating cortical blasts.     

Classification of temporal patterns in dynamic biological networks

A general method is presented to classify temporal patterns generated by rhythmic biological networks when synaptic connections and cellular properties are known. The method is discrete in nature and relies on algebraic properties of state transitions and graph theory. Elements of the set of rhythms generated by a network are compared using a metric that quantifies the functional differences among them. The rhythms are then classified according to their location in a metric space. Examples are given, and biological implications are discussed.     

Interaction between retinoic acid and vitamin D signaling pathways

The nuclear signaling pathways for retinoids and vitamin D differ in the specificity of the respective receptors for response elements. Two pathways for the action of both retinoic acid receptors (RARs) and vitamin D receptors (VDRs) have been identified, one being retinoid X receptor (RXR)-dependent and the other being RXR-independent. Moreover, RXRs were found to function as homodimers. In several steps we converted the retinoid specific response element of the human retinoic acid receptor beta promoter into the vitamin D/retinoic acid response element of the human osteocalcin promoter. We found that VDR homodimers only bind to the motif RGGTGA. The extended osteocalcin element also contains an imperfect direct repeat based on the motif RGGTGA spaced by three nucleotides, which is bound by RXR homodimers and activated by 9-cis-retinoic acid. The responsiveness of the osteocalcin element to all-trans-retinoic acid is mediated neither by RAR homodimers nor by RAR-RXR heterodimers. However, a VDR-RAR heterodimer binds to the osteocalcin response element and mediates activation by all-trans-retinoic acid. This heterodimer also binds to pure retinoid response elements, but it does not mediate activation by vitamin D alone. In combination with all-trans-retinoic acid, however, vitamin D enhances VDR-RAR heterodimer-mediated gene expression. This finding suggests a direct interaction between nuclear signaling by retinoic acid and vitamin D increasing the combinatorial possibilities for gene regulation by the nuclear receptors involved.     

Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways

In Drosophila, planar cell polarity (PCP) signaling is mediated by the receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). Wingless (Wg) signaling also requires Dsh and may utilize DFz2 as a receptor. Using a heterologous system, we show that Dsh is recruited selectively to the membrane by Fz but not DFz2, and this recruitment depends on the DEP domain but not the PDZ domain in Dsh. A mutation in the DEP domain impairs both membrane localization and the function of Dsh in PCP signaling, indicating that translocation is important for function. Further genetic and molecular analyses suggest that conserved domains in Dsh function differently during PCP and Wg signaling, and that divergent intracellular pathways are activated. We propose that Dsh has distinct roles in PCP and Wg signaling. The PCP signal may selectively result in focal Fz activation and asymmetric relocalization of Dsh to the membrane, where Dsh effects cytoskeletal reorganization to orient prehair initiation.     

Dissection of signaling pathways and cloning of new signal transducers in tyrosine kinase-induced pathways by genetic selection

We used genetic strategies which have been proven valuable to decipher signaling pathways in comparatively simple organisms such as Drosophila and Caenorhabditis elegans, to dissect signaling network activated by tyrosine kinases in mammals. The strategy was developed further towards a generally applicable expression cloning system to identify signal transducers in tyrosine kinase pathways. This system is based on the ability of downstream acting genes to rescue the transformation phenotype of partial loss-of-function mutants of BCR-ABL which still retain tyrosine kinase activity. Using this strategy we have previously shown that overexpression of c-Myc and Cyclin D1 can rescue a signaling defective SH2 mutant of BCR-ABL for transformation. In an unbiased approach to identify new compensating genes, a cDNA library was introduced by retroviral infection into fibroblasts which express the BCR-ABL SH2 mutant. CDNA clones, capable of rescuing the SH2 mutant for transformation should result in colony formation in soft agar. A PCR approach was used to recover these compensating genes from the genomic DNA of the transformed fibroblasts. Sequencing analysis of the initial cDNAs identified three known genes, the adapter molecule Shc, the kinases SPRK and p38 MAPK. These genes have been found to interact functionally with BCR-ABL for fibroblast and hematopoietic cell transformation. Currently, we are constructing and screening new libraries to identify novel genes which complement the BCR-ABL SH2 mutant. Our results demonstrate that this cloning approach is an effective means of identifying and characterizing signaling molecules that function in specific signaling pathways. This in turn may identify specific targets for mechanism-based therapeutic intervention to block altered signaling.     

Social senses: G-protein-coupled receptor signaling pathways in Dictyostelium discoideum

Incubation of phenol-induced cells of the yeast Candida maltosa SBUG 700 with mono- and dichlorophenols resulted in the formation of metabolites of the substrates and of further metabolites not related to the degradation pathway of the substrates. These additional compounds, identified as 4-hydroxyphenylacetic acid (4-HPA), phenylacetic acid (PA), indolylacetic acid (IA) and indolylethanol (i.e.) by means of HPLC and GC/MS, were not excreted in incubation experiments with glucose. The excretion of these metabolites of aromatic amino acid metabolism is not caused by toxic effects of the phenol derivatives, but seems to be a result of carbon and nitrogen starvation of yeast cells.     

Convergence and divergence of the signaling pathways for insulin and phosphoinositolglycans

Phosphoinositolglycan molecules isolated from insulin-sensitive mammalian tissues have been demonstrated in numerous in vitro studies to exert partial insulin-mimetic activity on glucose and lipid metabolism in insulin-sensitive cells. However, their ill-defined structures, heterogeneous nature, and limited availability have prohibited the analysis of the underlying molecular mechanism. Phosphoinositolglycan-peptide (PIG-P) of defined and homogeneous structure prepared in large scale from the core glycan of a glycosyl-phosphatidylinositol-anchored membrane protein from Saccharomyces cerevisiae has recently been shown to stimulate glucose transport as well as a number of glucose-metabolizing enzymes and pathways to up to 90% (at 2 to 10 microns) of the maximal insulin effect in isolated rat adipocytes, cardiomyocytes, and diaphragms (G. Müller et al., 1997, Endocrinology 138: 3459-3476). Consequently, we used this PIG-P for the present study in which we compare its intracellular signaling with that of insulin. The activation of glucose transport by both PIG-P and insulin in isolated rat adipocytes and diaphragms was found to require stimulation of phosphatidylinositol (PI) 3-kinase but to be independent of functional p70S6kinase and mitogen-activated protein kinase. The increase in glycerol-3-phosphate acyltransferase activity in rat adipocytes in response to PIG-P and insulin was dependent on both PI 3-kinase and p70S6kinase. This suggest that the signaling pathways for PIG-P and insulin to glucose transport and metabolism converage at the level of PI 3-kinase. A component of the PIG-P signaling pathway located up-stream of PI 3-kinase was identified by desensitization of isolated rat adipocytes for PIG-P action by combined treatment with trypsin and NaCl under conditions that preserved cell viability and the insulin-mimetic activity of sodium vanadate but completely blunted the insulin response. Incubation of the cells with either trypsin or NaCl alone was ineffective. The desensitized adipocytes were reconstituted for stimulation of lipogenesis by PIG-P by addition of the concentrated trypsin/salt extract. The reconstituted adipocytes exhibited 65-75% of the maximal PIG-P response and similar EC50 values for PIG-P (2 to 5 microns) compared with control cells. A proteinaceous N-ethylmaleimide (NEM)-sensitive component contained in the trypsin/salt extract was demonstrated to bind in a functional manner to the adipocyte plasma membrane of desensitized adipocytes via bipolar interactions. An excess of trypsin/salt extract inhibited PIG-P action in untreated adipocytes in a competitive fashion compatible with a receptor function for PIG-P of this protein. The presence of the putative PIG-P receptor protein in detergent-insoluble complexes prepared from isolated rat adipocytes suggests that caveolae/detergent-insoluble complexes of the plasma membrane may play a role in insulin-mimetic signaling by PIG-P. Furthermore, treatment of isolated rat diaphragms and adipocytes with PIG-P as well as with other agents exerting partially insulin-mimetic activity, such as PI-specific phospholipase C (PLC) and the sulfonylurea glimepiride, triggered tyrosine phosphorylation of the caveolar marker protein caveolin, which was apparently correlated with stimulation of lipogenesis. Strikingly, in adipocytes subjected to combined trypsin/salt treatment, PIG-P, PI-specific PLC, and glimepiride failed completely to provoke insulin-mimetic effects. A working model is presented for a signaling pathway in insulin-sensitive cells used by PIG(-P) molecules which involves GPI structures, the trypsin/salt- and NEM-sensitive receptor protein for PIG-P, and additional proteins located in caveolae/detergent-insoluble complexes.     

Effects of superoxide anions on endothelial Ca2+ signaling pathways

Although the involvement of free radicals in the development of endothelial dysfunction under pathological conditions, like diabetes and hypercholesterolemia, has been proposed frequently, there is limited knowledge as to how superoxide anions (O2-) might affect endothelial signal transduction. In this study, we investigated the effects of preincubation with the O2(-)-generating system xanthine oxidase/hypoxanthine (XO/HX) on mechanisms for Ca2+ signaling in cultured porcine aortic endothelial cells. Incubation of cells with XO/HX yielded increased intracellular Ca2+ release and capacitative Ca2+ entry in response to bradykinin and ATP in a time- and concentration-dependent manner. This effect was prevented by superoxide dismutase but not by the tyrosine kinase inhibitor tyrphostin A48. In addition, capacitative Ca2+ entry induced by the receptor-independent stimulus 2,5-di-(tert-butyl)-1,4-benzohydroquinone or thapsigargin was enhanced in O2(-)-exposed cells (+38% and +32%, respectively). Increased Ca2+ release in response to bradykinin in XO/HX-pretreated cells might be due to enhanced formation of inositol-1,4,5-trisphosphate (+140%). Exposure to XO/HX also affected other signal transduction mechanisms involved in endothelial Ca2+ signaling, such as microsomal cytochrome P450 epoxygenase and membrane hyperpolarization to Ca2+ store depletion with thapsigargin (+103% and +48%, respectively) and tyrosine kinase activity (+97%). A comparison of bradykinin-initiated intracellular Ca2+ release and thapsigargin-induced hyperpolarization with membrane viscosity modulated by XO/HX (decrease in viscosity) or cholesterol (increase in viscosity) reflected a negative correlation between bradykinin-initiated Ca2+ release and membrane viscosity. Because intracellular Ca2+ is a main regulator of endothelial vascular function, our data suggest that O2- anions are involved in regulation of the vascular endothelium.     

Dendritic cell survival and maturation are regulated by different signaling pathways

OBJECTIVE: Quantification of serum nucleotide pyrophosphohydrolase (NTPPHase) activity in healthy subjects and in patients with various rheumatic diseases or with quad/hemiplegia, hemodialysis, or renal transplant. METHODS: Colorimetric assay of enzyme activity in serum. RESULTS: Serum NTPPHase activity in 85 healthy subjects was independent of age or sex and was highly reproducible in each individual. The biologic and methodologic coefficients of variation were nearly identical. Elevated enzyme levels were found in sera from patients with osteoarthritis/spondylosis, calcium pyrophosphate dihydrate (CPPD) crystal deposition, scleroderma, fibromyalgia, or hemodialysis. Renal transplant patients receiving cyclosporine had the highest enzyme activity of any group, whereas transplant patients not taking this drug had normal levels. Histograms of values in all groups showed a normal distribution. CONCLUSION: Serum NTPPHase activity levels were significantly elevated in patients with degenerative arthritis whether or not CPPD crystals were present, in patients with either scleroderma or fibromyalgia, and in patients receiving hemodialysis therapy or taking cyclosporine.     

Emergent properties of neural systems: how focal molecular neurobiological alterations can affect behavior

Whereas the basic wiring diagram of the mammalian central nervous system (CNS) is genetically preprogramed, its fine tuning throughout different phases of infancy, childhood, and adulthood are highly experience dependent. The potential neurobiological mechanisms and behavioral effects of such experience-dependent neuroplasticity as a function of stage of development are outlined. A basic thesis of this paper is that mechanisms involved in neuronal learning and memory, such as long-term potentiation (LTP) and long-term depression (LTD), are used and reused not only in the sculpting of the CNS in the initial establishment of connections, but also again in the molding of personality and behavior based on experience. It is postulated that for higher order processes such as emotional memory, such neuroplasticity is occurring at increasingly larger numbers of synapses and cell assemblies with increasing mechanistic complexity and self-organization. Just as overexcitation or deprivation can profoundly affect the development of the visual system, it is postulated that similar phenomena exist in the neural substrates of emotional and cognitive development. In addition, a secondary and potentially more widespread series of ramifications are likely to occur in the higher order and integrative systems, such as secondary and tertiary cortical association areas and prefrontal cortex that become the ultimate integrators of emotion and experience, leading to subsequent actions and plans for the future. This process is by definition, plastic, and such remodeling is likely to take place not only at the level of the single synapse, but also at higher levels of network integration of which we currently have only the barest glimpse. Nonetheless, beginning to discuss the neurobiology of such self-organizing plastic systems may begin to change our conceptual approaches to psychopathology and open new avenues of therapeutics for the major psychiatric illnesses that are critically dependent on such higher order learning and memory mechanisms.