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D C Spray

Publications and source records attributed to D C Spray.

At least 55 records · Page 3Linked to original sources
PubMed1999

Voltage dependence of macroscopic and unitary currents of gap junction channels formed by mouse connexin50 expressed in rat neuroblastoma cells.

1. The macroscopic and single channel gating characteristics of connexin (Cx) 50 gap junction channels between pairs of N2A neuroblastoma cells transfected with mouse Cx50 DNA were investigated using the dual whole-cell voltage clamp technique. 2. The macroscopic junctional current (Ij) of Cx50-transfected cells decayed exponentially with time in response to transjunctional voltage (Vj) steps (time constant (tau) of approximately 4 s at a Vj of 30-40 mV and 100-200 ms at a Vj of 80-100 mV). The steady-state junctional conductance (gj) was well described by a two-state Boltzmann equation. The half-inactivation voltage (V0), the ratio of minimal to maximal gj (gmin/gmax) and the equivalent gating charge were +/- 37 mV, 0.21 and 4, respectively. 3. The conductance of single Cx50 channels measured using patch pipettes containing 130 mM CsCl was 220 +/- 13.1 pS (12 cell pairs). A prominent residual or subconductance state corresponding to 43 +/- 4. 2 pS (10 cell pairs) was also observed at large Vj s. 4. The relationship between channel open probability (Po) and Vj was well described by a Boltzmann relationship with parameters similar to those obtained for macroscopic gj (V0 = 34 mV, gating charge = 4.25, maximum P= 0.98). The ensemble average of single channel currents at Vj = 50 mV declined in a monoexponential manner (tau = 905 ms), a value similar to the decline of the macroscopic Ij of Cx50 channels at the same voltage. 5. Ion substitution experiments indicated that Cx50 channels have a lower permeability to anions than to cations (transjunctional conductance of KCl vs. potassium glutamate (gammaj, KCl/gammaj,KGlut), 1.2; 6 cell pairs). 6. The results have important implications for understanding the role of connexins in tissues where Cx50 is a major gap junction component, including the lens.

Animals↗
PubMed1999

Deficient assembly and function of gap junctions in Trf1, a trafficking mutant of the human liver-derived cell line HuH-7.

The Trf1 cell line, selected from the human hepatoma cell line HuH-7, manifests altered trafficking of various plasma membrane proteins. In particular, there is a striking loss of State 2 asialoglycoprotein receptors. This cell line is shown here to also manifest defects in function and assembly of gap junctions comprising connexin43 (Cx43). No alteration of Cx43 expression or phosphorylation was apparent. Nevertheless, immunostaining of Cx43 revealed that fewer and smaller gap junctions were present at appositional membrane areas in Trf1 cells as compared with parental HuH-7. This correlated with a significant attenuation in gap junction-mediated communication between Trf1 cells as demonstrated by markedly decreased dye transfer and their reduced ability to propagate mechanically evoked Ca(2+) waves. Isoelectric focusing (IEF) of Cx43 in HuH-7 cells indicated that the pIs of this protein were significantly lower than that predicted from its amino acid sequence; no differences in pI were evident in Cx43 from Trf1 cells and the HuH-7 cell line. The effects of the Trf1 mutation on assembly and function of gap junctions indicate that this mutation influences trafficking of Cx43. Connexins differ in several respects from other membrane proteins thus far analyzed in Trf1 mutants: gap junctions localize exclusively to the lateral cell surface; they are not glycoproteins; and they do not play a role in endocytic pathways. The disruption of trafficking of Cx43 by this mutation suggests that the Trf1 phenotype is a defect at a common point along the trafficking pathway of cell-surface proteins, irrespective of their ultimate destination on the cell surface or their glycosylation profile.

Asialoglycoprotein Receptor↗
PubMed1999

TPA induced expression and function of human connexin 26 by post-translational mechanisms in stably transfected neuroblastoma cells.

Connexin 26 (Cx26) has been proposed to be a tumor suppressor gene and its expression may modulate development, cell growth and differentiation in various tissues, including the brain. 12-O-tetradecanoylphorbol-13-acetate (TPA) may serve as either tumor promoter (in mammary gland amd skin) or as a differentiating agent (in neuroblastoma and leukemic cells) and may also modulate expression, function and phosphorylation of gap junctions. In this study, to determine the effects of TPA on Cx26 expression and its function in neuroblastoma, we transfected N2A mouse neuroblastoma cells (which are gap junction deficient) with the coding region of human Cx26 gene (which lacks TPA response elements) and examined the changes of expression and function of Cx26 following 10 nM TPA treatment. Individual clones of transfectants stably expressed distinct levels of exogenous Cx26 as judged by Northern and Western blots, immunocytochemistry and electrophysiological recordings. Cx26 channels displayed unitary conductances of about 140-155 pS. Increase of Cx26 expression following TPA treatment was markedly observed using immunocytochemistry and Western blots of membrane fractions although it was not detected in Northern or Western blots of whole cells. This increase in Cx26 expression in the plasma membrane was accompanied by an increase of function as evidenced in measurements of junctional conductance. These results suggest that induction of exogenous Cx26 in neuroblastoma cells by TPA treatment is controlled by post-translational mechanisms.

Animals↗
PubMed1999

A simple RT-PCR-based strategy for screening connexin identity.

Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx) proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s) can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain) that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins.

Animals↗
PubMed1998

Gap junction disappearance in astrocytes and leptomeningeal cells as a consequence of protozoan infection.

Trypanosoma cruzi and Toxoplasma gondii are protozoan parasites capable of causing infections of the nervous system. In order to determine effects of infection by these organisms on intercellular communication in the brain, dye coupling and connexin abundance and distribution were examined in leptomeningeal cells and astrocytes infected with T. cruzi or T. gondii. For both cell types infected with either type of protozoan parasite, intercellular diffusion of intracellularly injected Lucifer Yellow was dramatically reduced. Immunocytochemistry with antibodies specific for connexin43 (in astrocytes) or both connexin43 and connexin26 (for leptomeningeal cells) demonstrated that punctate gap junctional staining was much reduced in infected cells, although uninfected neighbors could display normal connexin abundance and distribution. Western blot analyses revealed that connexin43 abundance in both cell types infected with either parasite was similar to that in uninfected cells. Phosphorylation state of connexin43 (inferred from electrophoretic mobility of connexin43 isoforms) was not significantly affected by the infection process. Immunocytochemistry of whole brains from animals acutely infected with either parasite also showed a marked reduction in connexin43 expression. We conclude that infection of both types of brain cells with either protozoan parasite results in a loss of intercellular communication and organized gap junction plaques without affecting expression levels or posttranslational processing of gap junction proteins. Presumably, these changes in gap junction distribution result from altered targeting of the junctional protein to the plasma membrane, and/or from changes in assembly of subunits into functional channels.

Animals↗
PubMed1998

Changes in the properties of gap junctions during neuronal differentiation of hippocampal progenitor cells.

The cellular mechanisms that regulate progenitor cell lineage elaboration and maturation during embryonic development of the mammalian brain are poorly understood. Conditionally immortalized mouse hippocampal multipotent progenitor cells (MK31 cells) were found to be strongly coupled by gap junctions comprising connexin 43 (Cx43) during early neuronal ontogeny; the presence of this Cx type was confirmed by electrophysiological, molecular biological, and immunocytochemical assays. However, as progenitor cells underwent intermediate stages of neuronal differentiation under the influence of interleukin 7 (IL-7) alone or terminal differentiation after composite exposure to basic fibroblast growth factor, IL-7, and transforming growth factor alpha, coupling strength and the level of Cx43 expression declined. An additional population of junctional channels with distinct properties was detected at an intermediate stage of neuronal differentiation. Reverse transcription-PCR assays detected mRNA encoding Cx40 in IL-7-treated cells and Cx33 after both treatment conditions. Because functional channels in exogenous expression systems are not formed by pairing Cx40 with Cx43 or by pairing Cx33 with itself or additional connexins, these experimental observations raise the possibility that the progressive loss of coupling during differentiation of neural progenitor cells may involve downregulation of Cx43 coupled with potentiation of expression of Cx33 and Cx40. Furthermore, continued expression of Cx43 in differentiating neuroblasts could mediate intercellular communication between neuronal precursor cells and astrocytes by direct signaling via homotypic gap junction channels.

Animals↗
PubMed1998

Calcium waves between astrocytes from Cx43 knockout mice.

Gap junctions are regarded as the primary pathway underlying propagation of Ca2+ waves between astrocytes, although signaling through extracellular space may also contribute. Results obtained from astrocytes cultured from sibling Cx43 knockout (KO) and wild-type (WT) mice in six litters showed that Ca2+ waves propagated more slowly in Cx43 KO than in WT astrocytes; however, because this difference in velocity was only seen in conditions where cell confluence was higher in WT than KO astrocytes, it is attributable to differences in plating density. By contrast, density-independent differences were observed in the amplitudes of the Ca2+ responses (15% smaller in KO astrocytes) and efficacy of spread (to 14% fewer cells in KO astrocytes). Blockade of purinergic receptors with suramin reduced the velocities of the waves by 40% in WT and KO astrocytes and reduced the amplitudes by 20% and 6%, respectively. In the presence of heptanol, Ca2+ waves spread to only 30% of the cells, with a 70% reduced velocity and 30% reduced amplitude. It is concluded that the propagation of Ca2+ waves between astrocytes from Cx43 KO mice is not so greatly affected as expected by deletion of the major gap junction protein between these cells. The residual 5% coupling contributed by the additional connexins (Cx40, Cx45, and Cx46) expressed in KO astrocytes still suffices to provide a more substantial portion of Ca2+ wave propagation than does signaling through extracellular purinergic pathways. These studies demonstrate that, even with severely reduced junctional conductance, Cx43 KO astrocytes are capable of performing long-range Ca2+ wave signaling, perhaps preserving one mechanism critical to neural function.

Animals↗
PubMed1998

Decreased gap-junctional communication associated with segregation of the neuronal phenotype in the RT4 cell-line family.

RT4 is a family of cell lines derived from a rat peripheral neurotumor. The RT4 family consists of a multipotential stem-cell line that spontaneously gives rise to three derivative cell types, one glial and two neuronal. The three derivative cell types are capable of further lineage-specific maturation under appropriate culture conditions. Gap-junctional communication is postulated to be important during nervous-system development by allowing and/or controlling the transmission of both electrical current and signaling molecules, which may affect growth and differentiation. Our characterization of gap-junctional communication in the RT4 cell line family revealed that: (1) the glial-derivative and the stem-cell line were extensively coupled, while the two neuronal derivatives were significantly less coupled, and (2) all of the RT4 cell lines, including the stem-cell line, expressed Cx43 mRNA and protein, and the levels were generally consistent with the observed degree of functional coupling. These observations are consistent with data from in vivo studies and establish the RT4 cell line family as a potentially useful in vitro model system for understanding the role(s) of gap-junctional communication during differentiation in the peripheral nervous system.

Animals↗
PubMed1998

Structural changes in lenses of mice lacking the gap junction protein connexin43.

PURPOSE: To investigate the role of the gap junction protein connexin43 (Cx43), which is predominantly expressed in lens epithelial cells in the control of lens development and organization. METHODS: Newborn mice in which the Cx43 gene was disrupted by homologous recombination were used. Lenses from Cx43 (-/-) mice and wild-type littermates were processed by using 2% glutaraldehyde fixation for light and transmission electron microscopy and by freezing in liquid nitrogen for light and confocal microscopy of immunofluorescence in cryosections. RESULTS: In wild-type mice, Cx43 was immunolocalized to apical and lateral regions of lens epithelial cells and throughout the cornea, iris, ciliary body, and retina. In the bow, or equatorial, region of the lens, Cx43 disappeared gradually at the margins of the epithelial layer, whereas major intrinsic polypeptide, MP26, and alpha-crystallins were only detected in differentiated fiber cells. Ultrastructural studies revealed that epithelial cells and epithelial fiber cells were connected by large gap junctions. Lens fiber cells were closely apposed to apical boundaries of epithelial cells and apposed to one another along their entire lengths. In Cx43 (-/-) mice, epithelial cells were connected more loosely. The distribution of MP26 and alpha-crystallin in bow region fiber cells in Cx43 (-/-) lenses was not distinguishable from that in the lenses of wild-type mice. Cx46 and Cx50 were also expressed in superficial and cortical fiber cells, with similar distributions in Cx43 (-/-) and wild-type mice. However, organization of appositional membranes between lens fiber cells and between fiber and epithelial cells differed dramatically in the Cx43 (-/-) lens. In contrast to the close apposition of cells in lenses of normal mice, fiber cells in Cx43 (-/-) lenses were largely separated from apical surfaces of epithelial cells, and large vacuolar spaces were apparent between fiber cells, most prominently in deeper cortical regions. CONCLUSIONS: The normal differentiation of lens fiber cells in the bow region in lenses of Cx43 (-/-) mice, evidenced by similar distributions of Cx46, Cx50, MP26, and alpha-crystallin, suggests that the expression of Cx43 is not required for this process. However, these lenses exhibit grossly dilated extracellular spaces and intracellular vacuoles, indicative of early stages of cataract formation. These changes suggest that osmotic balance within the lens is markedly altered in Cx43 (-/-) animals, highlighting the importance of intercellular communication mediated by lens epithelial Cx43 gap junctions in the function of this tissue.

Animals↗
PubMed1998

Increased intercellular communication in mouse astrocytes exposed to hyposmotic shocks.

When exposed to 20% and 35%, but not to 50% hyposmotic solutions, mouse astrocytes recovered their volume within a few minutes, which coincided with the activation of nonjunctional conductances. Conductance of gap junctions between astrocyte pairs also increased after exposure to a 35% hyposmotic shock; however, this effect began at 3 min after the shock, when cells had partially recovered their initial volumes. During the first minute of exposure to 20% and 35% hyposmotic stimuli, there was a transient monophasic increase in intracellular calcium levels; exposure to 50% hyposmotic solution led to intracellular Ca2+ oscillations. The differences in time courses of nonjunctional conductance changes, Ca2+ alterations, and intercellular coupling suggest that distinct second messenger pathways are involved in each response. The velocity of mechanically evoked calcium waves propagated among the astrocytes increased at 7.5 min after 35% hyposmotic shock. This increase was not seen with 20% or 50% hyposmotic stimuli and is not ascribable to the increase in junctional conductance because it was blocked by suramin, a P2 purinergic receptor antagonist. Given that the transduction pathways activated during cell swelling (e.g., generation of phospholipases, phosphokinases, arachidonic acid) exert inhibitory effects on astrocytic gap junctions (Giaume and McCarthy, 1996), it is proposed that the increased junctional conductance during hyposmotic shock is due to increased number of channels, perhaps triggered by the initial Ca2+ signals (Dolmetsch et al., 1997). As a functional consequence of the increased coupling and enhanced extracellular propagation of Ca2+ waves, spread of signaling molecules throughout the glial network is expected to be significantly enhanced during hyposmotic stress. The increased intercellular communication between mouse astrocytes in response to hyposmotic challenge thus occurs via both gap junction-dependent and -independent mechanisms and presumably provides neuroprotective effects following nervous system injury.

Animals↗
PubMed1997

Phosphorylation of connexin43 and the regulation of neonatal rat cardiac myocyte gap junctions.

The functional state of gap junctions and the state of phosphorylation of connexin43 (Cx43), the major gap junction protein in rat heart, were evaluated in primary cultures of neonatal rat cardiocytes. Functional coupling was greatly reduced after treatment with staurosporine (ST), a protein kinase inhibitor. The ST-induced reduction in cell coupling was reversed by activation of protein kinase C (PKC) with 12-O-tetradecanoylphorbol 13-acetate (TPA). The cellular distribution of Cx43, as detected by immunofluorescence, was not grossly affected by either ST alone or ST plus TPA. Although immunoblot analysis did not detect significant changes in the relative amounts of the unphosphorylated and individual phosphorylated forms of Cx43 after each treatment, the level of 32P-incorporation into Cx43 of radiolabeled cells was significantly affected. Consistent with their known properties, treatment with ST reduced, and combined treatment with TPA and ST increased, the level of 32P-incorporation into Cx43. Two-dimensional tryptic phosphopeptide maps of 32P-labeled Cx43 indicated that a distinct subset of the phosphopeptides that are present under basal conditions were affected by ST or ST/TPA treatments, with TPA-induced phosphorylation occurring at the ST-sensitive sites. However, the ST/TPA-sensitive tryptic phosphopeptides did not comigrate with others that were derived from in vitro phosphorylation by PKC of a recombinant C-terminal Cx43 peptide (Cx43[243-382]). Although a PKC-dependent mechanism appears to be involved in the regulation of functional coupling between neonatal rat cardiocytes, PKC itself may not be the final mediator of Cx43 phosphorylation.

Animals↗
PubMed1997

The bystander effect exerted by tumor cells expressing the herpes simplex virus thymidine kinase (HSVtk) gene is dependent on connexin expression and cell communication via gap junctions.

To elucidate the role gap junctions play in the bystander effect, we examined the cytotoxic effect of herpes simplex virus thymidine kinase (HSVtk) modified tumor cells on gap junction communication-deficient tumor cells and their connexin transfectants. Communication competent Walker 256 tumor cells engineered to express the HSVtk gene (Walker-tk+) when cocultured with N2A mouse neuroblastoma and PC12 rat pheochromocytoma cells with absent endogenous junctional conductance showed no bystander cytotoxicity. Transfection of N2A cells with the rat connexin37 gene (5Q) and PC12 cells with the human connexin43 gene rendered them susceptible to bystander cell death. Additionally, communication-deficient N2A cells transfected with the HSVtk gene failed to exert a bystander effect, whereas N2A transfectants coexpressing the connexin37 and HSVtk genes (5Qtk+ cells) exerted bystander cytotoxicity on gap junction communication-competent 5Q but not on communication-deficient N2A cells in vitro. In vivo experiments also showed tumor growth inhibition of communication-competent 5Q but not communication-incompetent N2A cells by 5Qtk+ cells. In conclusion, these results indicate that in several cellular environments the bystander effect is dependent on connexin expression and gap junctional communication between HSVtk-positive and HSVtk-negative cells.

Animals↗
PubMed1997

Oligodendrocytes express gap junction proteins connexin32 and connexin45.

Oligodendrocytes, the myelin-forming glia of brain, are connected by gap junctions in situ and in culture. Cultured oligodendrocytes from adult bovine and porcine brains were studied using immunocytochemical, molecular, and electrophysiological techniques in order to characterize the gap junction types. The expression of connexin32 was substantiated by the detection of low, but significant, signals using connexin-specific probes in Northern and Western blot analyses. Connexin43, which comprises gap junctions in astrocytes, was not detectable in pure oligodendrocytic cultures; mRNAs of connexin40 and connexin37 and connexin26 were also not detected. By means of two specific antibodies directed to the recently cloned connexin45 and by RT-PCR we were able to identify this connexin as a second oligodendrocytic gap junction protein. Whole cell voltage clamp recording provided evidence for electrical coupling between pairs of cultured oligodendrocytes (mean junctional conductance 3.9 nS, n = 38 pairs) and intracellular Lucifer Yellow injection indicated that oligodendrocytes were usually only weakly dye coupled, with spread generally being restricted to nearest neighbors. Unitary conductances ranged from > 20 to < 150 pS with modes of distribution at about 100 to 120pS and 40 to 20 pS, respectively. These unitary conductances are consistent with the channel events expected for connexin32 and connexin45. The low degree of functional coupling between oligodendrocytes in vitro corresponds with the low levels of connexin32 and connexin45 messenger RNAs and protein expression.

Animals↗
PubMed1996

C-erbB2/neu transfection induces gap junctional communication incompetence in glial cells.

Astrocytes form functional networks that participate in active signaling in which external stimuli are generated and amplified in many of the same ways as in neurons. Gap junctions between astrocytes offer the structural avenue by which the electrical and metabolic signals are propagated from one cell to another. Little is known about the trafficking, assembly, and degradation mechanisms of the major astrocytic gap junction protein connexin43. We have studied a glial cell line transfected with the C-erbB2/neu oncogene (neu+), finding severe interruption of gap junctional communication after stable transfection. Evidence from Western blotting and phosphorylation studies showed that the processing of connexin43 to its higher phosphorylated isoforms is disturbed. Confocal laser imaging indicates that the major deficit in the neu+ cells is attributable to a lack in plaque assembly of connexin43. Because the neu+ cells also lack N-CAM proteins and because work from others has indicated a close relationship between communication competence and constitutive CAM expression, our data suggest that expression of C-erbB2/neu oncogene alters cell-cell association via CAM proteins, which thereby affects gap junction plaque assembly and appropriate phosphorylation of connexin43.

Animals↗
PubMed1996

Are there functional gap junctions or junctional hemichannels in macrophages?

The existence of functional gap junctions in migratory cells of the immune system is a controversial issue. In this report, we have focused on one particular cell type, namely the macrophages, because connexin-43, a protein that forms gap junctions, has been described in peritoneal macrophages and a macrophage cell line (J774), by Northern and Western blot analysis. To test whether these cell types expressed functional gap junctions, we assayed dye coupling by intracellular injection of Lucifer Yellow. We observed that nonstimulated macrophages are not coupled among themselves and did not form functional gap junctions with an epithelial cell line, which expresses functional gap junctions formed by connexin-43. Dye coupling was also not detected between macrophages previously activated by lipopolysaccharide or interferon-gamma. We further examined the presence of functional coupling using the more sensitive technique of dual whole cell patch-clamp, and again, did not find electrical coupling between macrophages, consistent with the dye microinjection data. We also examined the possible presence of hemigap junction channels activated by extracellular adenosine triphosphate (ATP) using a dye uptake assay and the whole cell patch-clamp technique. Conditions expected to close gap junction hemichannels (exposure to octanol and low intracellular pH) did not decrease ATP-induced Lucifer Yellow uptake, whereas conditions expected to increase hemichannel opening either did not affect ATP permeabilization (dibutyryl adenosine monophosphate) or decreased it (zero extracellular CA+2). Finally, in experiments using resident macrophages derived from conexin-43 knockout mice, we observed ATP induced dye uptake. Our experimental data thus indicate that macrophages in vitro do not form functional gap junctions and that the permeability pathway activated by extracellular ATP is not formed by a hemigap junction channel.

1-Octanol↗