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D C Spray

Publications and source records attributed to D C Spray.

At least 37 records · Page 2Linked to original sources
PubMed2000

KATP channels regulate mitogenically induced proliferation in primary rat hepatocytes and human liver cell lines. Implications for liver growth control and potential therapeutic targeting.

To determine whether K(ATP) channels control liver growth, we used primary rat hepatocytes and several human cancer cell lines for assays. K(ATP) channel openers (minoxidil, cromakalim, and pinacidil) increased cellular DNA synthesis, whereas K(ATP) channel blockers (quinidine and glibenclamide) attenuated DNA synthesis. The channel inhibitor glibenclamide decreased the clonogenicity of HepG2 cells without inducing cytotoxicity or apoptosis. To demonstrate the specificity of drugs for K(+) channels, whole-cell patch-clamp recordings were made. Hepatocytes revealed K(+) currents with K(ATP) channel properties. These K(+) currents were augmented by minoxidil and pinacidil and attenuated by glibenclamide as well as tetraethylammonium, in agreement with established responses of K(ATP) channels. Reverse transcription of total cellular RNA followed by polymerase chain reaction showed expression of K(ATP) channel-specific subunits in rat hepatocytes and human liver cell lines. Calcium fluxes were unperturbed in glibenclamide-treated HepG2 cells and primary rat hepatocytes following induction with ATP and hepatocyte growth factor, respectively, suggesting that the effect of K(ATP) channel activity upon hepatocyte proliferation was not simply due to indirect modulation of intracellular calcium. The regulation of mitogen-related hepatocyte proliferation by K(ATP) channels advances our insights into liver growth control. The findings have implications in mechanisms concerning liver development, regeneration, and oncogenesis.

ATP-Binding Cassette Transporters↗
PubMed2000

Evidence for secretory pathway localization of a voltage-dependent anion channel isoform.

Voltage-dependent anion channels (VDACs) are pore-forming proteins (porins) that form the major pathway for movement of adenine nucleotides through the outer mitochondrial membrane. Electrophysiological studies indicate that VDAC-like channel activity is also prevalent in the cell membranes of many mammalian cells. However, the multitopological localization of porins outside the mitochondrion has remained an extremely controversial issue. Herein, we show that usage of two alternative first exons of the murine VDAC-1 gene leads to expression of two porins differing within their N termini. One porin (plasmalemmal VDAC-1) harboring a hydrophobic leader peptide is primarily targeted through the Golgi apparatus to the cell membrane. In contrast, the second isoform lacking the N-terminal leader (mitochondrial VDAC-1) is translocated more efficiently into the outer mitochondrial membrane. Thus, our data provide unique genetic evidence in favor of a multitopological localization of a mitochondrial porin.

Animals↗
PubMed2000

Intercellular communication in spinal cord astrocytes: fine tuning between gap junctions and P2 nucleotide receptors in calcium wave propagation.

Electrophysiological properties of gap junction channels and mechanisms involved in the propagation of intercellular calcium waves were studied in cultured spinal cord astrocytes from sibling wild-type (WT) and connexin43 (Cx43) knock-out (KO) mice. Comparison of the strength of coupling between pairs of WT and Cx43 KO spinal cord astrocytes indicates that two-thirds of total coupling is attributable to channels formed by Cx43, with other connexins contributing the remaining one-third of junctional conductance. Although such a difference in junctional conductance was expected to result in the reduced diffusion of signaling molecules through the Cx43 KO spinal cord syncytium, intercellular calcium waves were found to propagate with the same velocity and amplitude and to the same number of cells as between WT astrocytes. Measurements of calcium wave propagation in the presence of purinoceptor blockers indicate that calcium waves in Cx43 KO spinal cord astrocytes are mediated primarily by extracellular diffusion of ATP; measurements of responses to purinoceptor agonists revealed that the functional P2Y receptor subtype is shifted in the Cx43 KO astrocytes, with a markedly potentiated response to ATP and UTP. Thus, the reduction in gap junctional communication in Cx43 KO astrocytes leads to an increase in autocrine communication, which is a consequence of a functional switch in the P2Y nucleotide receptor subtype. Intercellular communication via calcium waves therefore is sustained in Cx43 null mice by a finely tuned interaction between gap junction-dependent and independent mechanisms.

Adenosine Triphosphate↗
PubMed2000

Properties of gap junction channels formed by Cx46 alone and in combination with Cx50.

Gap junctions formed of connexin46 (Cx46) and connexin50 (Cx50) in lens fiber cells are crucial for maintaining lens transparency. We determined the functional properties of homotypic Cx46, heterotypic Cx46/Cx50, and heteromeric Cx46/Cx50 channels in a communication-deficient neuroblastoma (N2A) cell line, using dual whole-cell recordings. N2A cultures were stably and/or transiently transfected with Cx46, Cx50, and green fluorescent protein (EGFP). The macroscopic voltage sensitivity of homotypic Cx46 conformed to the two-state model (Boltzmann parameters: G(min) = 0.11, V(0) = +/- 48.1 mV, gating charge = 2). Cx46 single channels showed a main-state conductance of 140 +/- 8 pS and multiple subconductance states ranging from < or =10 pS to 60 pS. Conservation of homotypic properties in heterotypic Cx46/Cx50 cell pairs allowed the determination of a positive relative gating polarity for the dominant gating mechanisms in Cx46 and Cx50. Observed gating properties were consistent with a second gating mechanism in Cx46 connexons. Moreover, rectification was observed in heterotypic cell pairs. Some cell pairs in cultures simultaneously transfected with Cx46 and Cx50 exhibited junctional properties not observed in other preparations, suggesting the formation of heteromeric channels. We conclude that different combinations of Cx46 and Cx50 within gap junction channels lead to unique biophysical properties.

Animals↗
PubMed2000

Connexin43 null mice reveal that astrocytes express multiple connexins.

The gap junction protein connexin43 (Cx43) is the primary component of intercellular channels in cardiac tissue and in astrocytes, the most abundant type of glial cells in the brain. Mice in which the gene for Cx43 is deleted by homologous recombination die at birth, due to profound hypertrophy of the ventricular outflow tract and stenosis of the pulmonary artery. Despite this significant cardiovascular abnormality, brains of connexin43 null [Cx43 (-/-)] animals are shown to be macroscopically normal and to display a pattern of cortical lamination that is not detectably different from wildtype siblings. Presence of Cx40 and Cx45 in brains and astrocytes cultured from both Cx43 (-/-) mice and wildtype littermates was confirmed by RT-PCR, Northern blot analyses and by immunostaining; Cx46 was detected by RT-PCR and Northern blot analyses. Presence of Cx26 in astrocyte cultures was indicated by RT-PCR and by Western blot analysis, although we were unable to resolve whether it was contributed by contaminating cells; Cx30 mRNA was detected by Northern blot in long term (2 weeks) but not fresh cultures of astrocytes. These studies thus reveal that astrocyte gap junctions may be formed of multiple connexins. Presumably, the metabolic and ionic coupling provided by these diverse gap junction types may functionally compensate for the absence of the major astrocyte gap junction protein in Cx43 (-/-) mice, providing whatever intercellular signaling is necessary for brain development and cortical lamination.

Animals↗
PubMed2000

Gap junctions in the nervous system.

Synapses are classically defined as close connections between two nerve cells or between a neuronal cell and a muscle or gland cell across which a chemical signal (i.e., a neurotransmitter) and/or an electrical signal (i.e., current-carrying ions) can pass. The definition of synapse was developed by Charles Sherrington and by Ramon y Cajal at the beginning of this century and refined by John Eccles and Bernard Katz 50 years later; in this collection of papers, the definition of synapses is discussed further in the chapter by Mike Bennett. who provided the first functional demonstration of electrical transmission via gap junction channels between vertebrate neurons. As is evidenced by the range of topics covered in this issue, research dealing with gap junctions in the nervous system has expanded enormously in the past decade, major findings being that specific cell types in the brain expresses specific types of connexins and that expression patterns coincide with tissue compartmentalization and function and that these compartments change during development.

Animals↗
PubMed2000

Temporal expression of neuronal connexins during hippocampal ontogeny.

Communication through gap junction channels provides a major signaling mechanism during early brain histogenesis, a developmental time during which neural progenitor cells are inexcitable and do not express ligand-gated channel responses to the major CNS neurotransmitters. Expression of different gap junction types during neurogenesis may therefore define intercellular pathways for transmission of developmentally relevant molecules. To better understand the molecular mechanism(s) by which growth and differentiation of neurons are modulated by gap junction channels, we have been examining the developmental effects of a specific set of cytokines on differentiation and gap junction expression in a conditionally immortalized mouse embryonic hippocampal neuronal progenitor cell line (MK31). When multipotent MK31 cells are in an uncommitted state, they uniformly express the neuroepithelial intermediate filament class VI marker, nestin, are strongly coupled by gap junctions composed of connexin43 (Cx43) and express connexin45 (Cx45) at the mRNA level. As these cells undergo neuronal lineage commitment and exit from cell cycle, they begin to express the early neurofilament marker, NF66, and coupling strength and expression of Cx43 begin to decline with concurrent expression of other connexin proteins, including Cx26, Cx33, Cx36, Cx40 and Cx45. Terminal neuronal differentiation is heralded by the expression of more advanced neurofilament proteins, increased morphologic maturation, the elaboration of inward currents and action potentials that possess mature physiological properties, and changing profiles of expression of connexin subtypes, including upregulation of Cx36 expression. These important developmental transitions are regulated by a complex network of cell cycle checkpoints. To begin to examine the precise roles of gap junction proteins in traversing these developmental checkpoints and in thus regulating neurogenesis, we have focused on individual members of two classes of genes involved in these seminal events: ID (inhibitor of differentiation)-1 and GAS (growth arrest-specific gene)5. When MK31 cells were maintained in an uncommitted state, levels of ID-1 mRNA were high and GAS5 transcripts were essentially undetectable. Application of cytokines that promote neuronal lineage commitment and cell cycle exit resulted in down-regulation of ID-1 and upregulation of GAS5 transcripts, whereas additional cytokine paradigms that promoted terminal neuronal differentiation resulted in the delayed down-regulation of GAS5 mRNA. Stable MK31 transfectants were generated for ID-1 and GAS5. In basal conditions, cellular proliferation was enhanced in the ID-1 transfectants and inhibited in the GAS5 transfectants when compared with control MK31 cells. When cytokine-mediated neurogenesis was examined in these transfected cell lines, constitutive expression of ID-1 inhibited and constitutive expression of GAS5 enhanced initial and terminal stages of neuronal differentiation, with evidence that terminal neuronal maturation in both transfectant lines was associated with decreased cellular viability, possibly due to the presence of conflicting cell cycle-associated developmental signals. These experimental reagents will prove to be valuable experimental tools to help define the functional interrelationships between changing profiles of connexin protein expression and cell cycle regulation during neuronal ontogeny in the mammalian brain. The present review summarizes the current state of research involving the temporal expression of such connexin types in differentiating hippocampal neurons and speculates on the possible role of these intercellular channels in the development and plasticity of the nervous system. In addition, we describe the functional properties and expression pattern of the newly discovered neuronal-specific gap junctional protein, Cx36, in the developing mouse fetal hippocampus and in the rat retina and brain.

Animals↗
PubMed2000

Gap junctions: the "kiss of death" and the "kiss of life".

Cells expressing herpes simplex-thymidine kinase (HSV-tk) can be killed "in vitro" within 5 days of treatment with 20 microM ganciclovir (GCV) and transmit this toxicity to adjacent cells lacking HSV-tk; this phenomenon was termed "bystander effect" or "kiss of death". On testing a large number of cell lines in vitro, a wide range of sensitivity to GCV-mediated bystander killing has been reported. Although intercellular transfer of GCV metabolites through gap junction channels seems to be a likely mechanism for the "kiss of death", some studies suggest that other pathways may contribute to induced apoptosis of neighboring cells. To further investigate the mechanism underlying cell death mediated by HSV-tk and to evaluate the efficacy of gap junction channels formed by different connexins in this process, we have stably transfected a virtually uncoupled mouse neuroblastoma cell line (N2A cells) with different connexin-types expressed by neural cells (Cx32, Cx37, Cx40, Cx43) and co-cultured these cells with N2A cells stably transfected with Cx37 and HSV-tk. Here, we confirm our previous studies and those of others that the extent of cell death and sensitivity to GCV depend on the degree of connexin expression in transfectants. Further, we show that the bystander effect also depends on which connexin is expressed; reported disparities regarding the extent of GCV-mediated cellular apoptosis are likely due both to the degree of functional coupling and the type of connexin expressed. These results support the notion that gap junction hemichannels formed of certain connexins are more likely than others to pair functionally with Cx37, and suggest co-transfection strategies that might prove effective in sensitizing tumor cell populations to GCV. In addition, potential applications are discussed for use of the "good Samaritan effect", a mechanism by which bystander cells have been suggested to prevent cytotoxicity.

Animals↗
PubMed2000

Slow intercellular Ca(2+) signaling in wild-type and Cx43-null neonatal mouse cardiac myocytes.

Focal mechanical stimulation of single neonatal mouse cardiac myocytes in culture induced intercellular Ca(2+) waves that propagated with mean velocities of approximately 14 micrometer/s, reaching approximately 80% of the cells in the field. Deletion of connexin43 (Cx43), the main cardiac gap junction channel protein, did not prevent communication of mechanically induced Ca(2+) waves, although the velocity and number of cells communicated by the Ca(2+) signal were significantly reduced. Similar effects were observed in wild-type cardiac myocytes treated with heptanol, a gap junction channel blocker. Fewer cells were involved in intercellular Ca(2+) signaling in both wild-type and Cx43-null cultures in the presence of suramin, a P(2)-receptor blocker; blockage was more effective in Cx43-null than in wild-type cells. Thus gap junction channels provide the main pathway for communication of slow intercellular Ca(2+) signals in wild-type neonatal mouse cardiac myocytes. Activation of P(2)-receptors induced by ATP release contributes a secondary, extracellular pathway for transmission of Ca(2+) signals. The importance of such ATP-mediated Ca(2+) signaling would be expected to be enhanced under ischemic conditions, when release of ATP is increased and gap junction channels conductance is significantly reduced.

Adenosine Triphosphate↗
PubMed2000

Wnt-1 regulation of connexin43 in cardiac myocytes.

Gap junction channels composed of connexin43 (Cx43) are essential for normal heart formation and function. We studied the potential role of the Wnt family of secreted polypeptides as regulators of Cx43 expression and gap junction channel function in dissociated myocytes and intact hearts. Neonatal rat cardiomyocytes responded to Li(+), which mimics Wnt signaling, by accumulating the effector protein beta-catenin and by inducing Cx43 mRNA and protein markedly. Induction of Cx43 expression was also observed in cardiomyocytes cocultured with Rat-2 fibroblasts or N2A neuroblastoma cells programmed to secrete bioactive Wnt-1. By transfecting a Cx43 promoter-reporter gene construct into cardiomyocytes, we demonstrated that the inductive effect of Wnt signaling was transcriptionally mediated. Enhanced expression of Cx43 increased cardiomyocyte cell coupling, as determined by Lucifer Yellow dye transfer and by calcium wave propagation. Conversely, in a transgenic cardiomyopathic mouse model that exhibits ventricular arrhythmias and gap junctional remodeling, beta-catenin and Cx43 expression were downregulated concordantly. In response to Wnt signaling, the accumulating Cx43 colocalized with beta-catenin in the junctional membrane; moreover, forced expression of Cx43 in cardiomyocytes reduced the transactivation potential of beta-catenin. These findings demonstrate that Wnt signaling is an important modulator of Cx43-dependent intercellular coupling in the heart, and they support the hypothesis that dysregulated signaling contributes to altered impulse propagation and arrhythmia in the myopathic heart.

Animals↗
PubMed2000

Gap junctions in the cardiovascular and immune systems.

Gap junctions are clusters of intercellular channels directly connecting the cytoplasm of adjacent cells. These channels are formed by proteins named connexins and are present in all metazoan organisms where they serve diverse functions ranging from control of cell growth and differentiation to electric conduction in excitable tissues. In this overview we describe the presence of connexins in the cardiovascular and lympho-hematopoietic systems giving the reader a summary of the topics to be covered throughout this edition and a historical perspective of the discovery of gap junctions in the immune system.

Animals↗
PubMed1999

Inhibition of endothelial cell migration, intercellular communication, and vascular tube formation by thromboxane A(2).

The eicosanoid thromboxane A(2) (TXA(2)) is released by activated platelets, monocytes, and the vessel wall and interacts with high affinity receptors expressed in several tissues including endothelium. Whether TXA(2) might alter endothelial migration and tube formation, two determinants of angiogenesis, is unknown. Thus, we investigated the effect of the TXA(2) mimetic [1S-(1alpha, 2beta(5Z),3alpha(1E,3R), 4alpha]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-o xab icyclo- [2.2.1]heptan-2-yl]-5'-heptenoic acid (IBOP) on human endothelial cell (HEC) migration and angiogenesis in vitro. IBOP stimulation inhibited HEC migration by 50% and in vitro capillary formation by 75%. These effects of IBOP were time- and concentration-dependent with an IC(50) of 25 nM. IBOP did not affect integrin expression or cytoskeletal morphology of HEC. Since gap junction-mediated intercellular communication increases in migrating HEC, we determined whether IBOP might inhibit coupling or connexin expression in HEC. IBOP reduced the passage of microinjected dyes between HEC by 50%, and the effects of IBOP on migration and tube formation were mimicked by the gap junction inhibitor 18beta-glycyrrhetinic acid (1 microM) with a similar time course and efficacy. IBOP (24 h) did not affect the expression or phosphorylation of connexin 43 in whole HEC lysates. Immunohistologic examination of HEC suggested that IBOP may impair functional coupling by altering the cellular distribution of gap junctions, leading to increased connexin 43 internalization. Thus, this finding that TXA(2) mimetics can prevent HEC migration and tube formation, possibly by impairing intercellular communication, suggests that antagonizing TXA(2) signaling might enhance vascularization of ischemic tissue.

Bridged Bicyclo Compounds, Heterocyclic↗
PubMed1999

Induction of tight junctions in human connexin 32 (hCx32)-transfected mouse hepatocytes: connexin 32 interacts with occludin.

Small gap junction plaques are associated with tight junction strands in some cell types including hepatocytes and it is thought that they may be closely related to tight junctions and the establishment of cell polarity. In order to examine roles of gap junctions in regulating expression and structure of tight junctions, we transfected human Cx32 cDNA into immortalized mouse hepatocytes (CHST8 cells) which lack endogenous Cx32 and Cx26. Immunocytochemistry revealed that endogenous integral tight junction protein occludin was strongly localized and was colocalized with Cx32 at cell borders in transfectants, whereas neither was detected in parental cells. In Northern blots, mRNAs encoding occludin and the other integral tight junction proteins, claudin-1 and -2, were induced in the transfectants compared to parental cells. In Western blots, occludin protein was increased in the transfectants compared to parental cells, and binding of occludin to Cx32 protein was demonstrated by immunoprecipitation. In freeze fracture of the transfectants, tight junction strands were more numerous and complex compared to parental cells, and small gap junction plaques appeared within induced tight junction strands. Nevertheless, no change in barrier function of tight junctions was observed. These results indicate that in hepatocytes, gap junction, and tight junction expression are closely coordinated, and that Cx32 may play a role in regulating occludin expression.

Animals↗
PubMed1999

Functional properties of channels formed by the neuronal gap junction protein connexin36.

The expression and functional properties of connexin36 (Cx36) were examined in two communication-deficient cell lines (N2A-neuroblastoma and PC-12 cells) transfected with Cx36 and in hippocampal neurons that express the connexin endogenously. Transfected cells expressed the expected 2.9 kb Cx36 transcript and Cx36 immunoreactivity, whereas nontransfected cells were devoid of Cx36. The relationship between steady-state junctional conductance (g(j)) and transjunctional voltage was well described by a two-state Boltzmann equation. The half-inactivation voltage (V(0)), the ratio of minimal to maximal g(j) (g(min)/g(max)), and the equivalent gating charge were +/- 75 mV, 0.55, and 1.75, respectively, indicating that Cx36 exhibits very low voltage sensitivity. Conductance of single Cx36 channels measured with patch pipettes containing 130 mM CsCl was 10-15 pS (n = 15 cell pairs); despite this low unitary conductance, Cx36 channels were permeable to the dye Lucifer yellow. Hippocampal neurons expressed Cx36 both in vivo and in culture. The electrophysiological properties of channels in cultured hippocampal neurons were similar to those of the channels expressed by the transfected cell lines, and the neuronal channels were similarly permeable to Lucifer yellow. The unique combination of weak voltage sensitivity, small unitary conductance, and permeation by anions as large as second messenger molecules endows Cx36 gap junction channels with properties well suited for mediating flexible electrical and biochemical interactions between neurons.

Animals↗
PubMed1999

IL-1beta differentially regulates calcium wave propagation between primary human fetal astrocytes via pathways involving P2 receptors and gap junction channels.

In mammalian astrocytes, calcium waves are transmitted between cells via both a gap junction-mediated pathway and an extracellular, P2 receptor-mediated pathway, which link the cells into a syncytium. Calcium waves in astrocytes have also been shown to evoke calcium transients in neurons, and activity in neurons can elicit calcium waves in astrocytes. In this study, we show that in primary human fetal astrocytes, the P2 receptor-mediated and gap junction-mediated pathways are differentially regulated by the cytokine IL-1beta. Confocal microscopy of astrocytes loaded with Indo-1 demonstrated that intercellular calcium wave transmission in IL-1beta-treated cultures was potentiated compared with controls. However, transmission of calcium waves via the gap junction-mediated pathway was strikingly reduced. The major component of functional gap junctions in human fetal astrocytes was demonstrated to be connexin43 (Cx43), and there was a marked reduction of junctional conductance, loss of dye coupling, loss of Cx43 protein, and down-regulation of Cx43 mRNA expression after IL-1beta treatment of cultures. Conversely, transmission of calcium waves via the P2 receptor-mediated pathway was potentiated in IL-1beta-treated cultures compared with controls. This potentiation was associated with an increase in the number of cells responsive to UTP, and with a transient increase in expression of the P2Y(2) purinoceptor mRNA. Because in inflammatory conditions of the human central nervous system IL-1beta is produced both by resident glia and by invading cells of the immune system, our results suggest that inflammatory events may have a significant impact on coordination of astrocytic function and on information processing in the central nervous system.

Astrocytes↗
PubMed1999

Gap-junctional coupling between neurons and astrocytes in primary central nervous system cultures.

Gap-junctional communication between neurons and astrocytes dissociated from rat brain was identified in culture by using dye-transfer assays and electrophysiological measurements. Cell types were identified by using antibodies against beta-tubulin III, glial fibrillary acidic protein, and 2',3'-cyclic-nucleotide phosphohydrolase, which are antigenic determinants of neurons, astroglia, and oligodendrocytes, respectively. Dye coupling was examined as a function of time after dissociated embryonic brain cells were plated onto confluent monolayers of postnatal astrocytes by intracellularly injecting the fluorochrome Lucifer yellow. Coupling of neurons to the astrocytic monolayer was most frequent between 48 h and 72 h in culture and declined over the next 4 days. This gradual uncoupling was accompanied by progressive neuronal maturation, as indicated by morphological measurements in camera lucida drawings. Dye spread was abolished reversibly by octanol, an agent that blocks gap junction channels in other systems. Double whole-cell voltage-clamp measurements confirmed the presence of heterocellular electrical coupling in these cocultures. Coupling was also seen between neurons and astrocytes in cocultures of cells dissociated from embryonic cerebral hemispheres but was rarely detectable in cocultures of postnatal brain cells. These data strongly suggest that junctional communication may provide metabolic and electrotonic interconnections between neuronal and astrocytic networks at early stages of neural development and that such interactions are weakened as differentiation progresses.

Animals↗