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D C Spray

Publications and source records attributed to D C Spray.

At least 91 records · Page 5Linked to original sources
PubMed1995

Transforming growth factor-beta 1 and forskolin modulate gap junctional communication and cellular phenotype of cultured Schwann cells.

Following peripheral nerve injury, Schwann cells undergo a series of cellular alterations that are thought to assist the regenerative process. Some of these changes are stimulated by the local release of cytokines and mitogenic factors. To test the hypothesis that cytokine regulation of gap junctional communication between cells helps to coordinate Schwann cell responses, cultured rat Schwann cells, from sciatic nerve, were utilized to study phasic changes induced by transforming growth factor-beta 1 (TGF beta 1), a cytokine released after nerve injury, or forskolin in combination with bovine pituitary extract (F-BPE), known for its mitogenic effects in vitro. In mitotically quiescent cultures, TGF beta 1 significantly decreased both electrical and dye coupling mediated by gap junctions. Single-channel analysis revealed that cultured Schwann cells expressed gap junctions with two distinct channel sizes of about 26 pS and 44 pS. TGF beta 1 treatment reduced coupling due to both populations of channels. Exposure to TGF beta 1 had a minimal effect on proliferation but significantly altered cellular morphology; cell bodies became flattened with multipolar processes within 72 hr. Additionally, immunolabeling for both low-affinity nerve growth factor receptor (L-NGFR) and glial fibrillary acidic protein (GFAP) were reduced, suggesting increased differentiation. In contrast, treatment with F-BPE significantly enhanced both electrical and dye coupling and stimulated Schwann cell proliferation. Additionally, cell bodies became more rounded with polarized, cytoplasmic processes contiguously aligned with adjacent cells. F-BPE reduced immunolabeling for L-NGFR but increased expression of both GFAP and the major peripheral myelin protein, P0. These data indicate that TGF beta 1 and/or F-BPE induce phenotypic changes in Schwann cells, including the coordinated regulation of proliferation and modulation of intercellular communication via gap junctions. Such mechanisms may underlie phasic responses that orchestrate recovery from nerve injury, indicating that Schwann cell gap junctions may be critical for peripheral nerve function.

Animals↗
PubMed1994

Human fetal neurons in culture: intercellular communication and voltage- and ligand-gated responses.

We established high density primary cultures of neural cells from dissociated second trimester human fetal brains using a novel spin seeding method. Under our culture conditions, the majority of the cells exhibited neuronal phenotypes as evidenced by morphological criteria, immunoreactivity to the 66 kDa neurofilament protein and expression of TTX-sensitive Na+ channels and cell excitability. These cultures were enriched in glutamic acid decarboxylase (GAD), the synthetic enzyme of the neurotransmitter GABA, and responded with Ca2+ influx to acute application of glutamate. Interestingly, the human fetal neurons in culture did not express either dopaminergic or cholinergic phenotypes. In addition, the population of neurons obtained express a high incidence of gap junction-mediated intercellular communication. These studies provide evidence that functional neuronal properties arise early during prenatal development in humans and offer the potential to evaluate pharmacological agents on primary human neurons.

Brain↗
PubMed1994

Cloning and in situ localization of a brain-derived porin that constitutes a large-conductance anion channel in astrocytic plasma membranes.

We have cloned a protein from bovine brain, brain-derived voltage-dependent anion channel 1 (BR1-VDAC), that is identical to a recently sequenced plasmalemmal-bound porin from human lymphocytes. mRNA hybridization indicates that BR1-VDAC is widely distributed throughout nervous and nonnervous tissues. In situ localization substantiated that the BR1-VDAC is associated with the plasmalemma of astrocytes. A monoclonal antibody that recognizes the N terminus of the BR1-VDAC protein completely blocks an astrocytic high-conductance anion channel that has electrophysiological similarities with the mitochondrial VDAC. Since the high-conductance anion channel in astrocytes has been shown to respond to hypoosmotic solutions, its molecular identification provides the basis for a better understanding of volume regulation in brain tissue.

Animals↗
PubMed1994

mRNAs encoding muscarinic and substance P receptors in cultured sympathetic neurons are differentially regulated by LIF or CNTF.

Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) have previously been shown to regulate neuronal choice of neurotransmitter. In this present study, these factors were shown to specifically and differentially regulate levels of both muscarinic (subtypes m1, m2, m3, m4, and m5) and substance P receptor (SPR) mRNAs in sympathetic neurons of the rat superior cervical ganglion (SCG) using solution hybridization/RNase protection analysis. In vivo, neonatal rat SCG expressed predominantly m2 (10.31 +/- 0.43 pg mRNA/micrograms total RNA) and some m1 (1.54 +/- 0.84 pg/microgram) muscarinic receptor mRNA, which increased developmentally to adult levels (m2 mRNA levels being 60% higher than those in neonates). By contrast, m3, m4, and m5 subtype mRNAs were much less abundant at all time points measured. A similar developmental regulation was found in dissociated SCG neurons in vitro. After 16 days in culture, m2 mRNA increased 334% to 15.76 +/- 0.68 pg/microgram, while m1 mRNA changed little (2.03 +/- 1.00 pg/microgram). However, LIF or CNTF treatment (5 ng/ml, 14 days) in sister cultures completely blocked this developmental increase. Further, LIF treatment blocked the normal muscarinic receptor-mediated increase in intracellular calcium (fura-2 imaging), indicating a functional change in receptor phenotype. By contrast, levels of SPR mRNA, which were low in untreated cultures (0.037 +/- 0.025 pg SPR mRNA/microgram total RNA), were elevated by LIF or CNTF treatment, to 0.866 +/- 0.034 pg/microgram and 0.662 +/- 0.148 pg/microgram, respectively. These observations indicate that muscarinic and SPR receptor expression are differentially regulated by the same factors in SCG neurons and that neuronal choice of receptor phenotype may be, at least in part, specifically regulated by cytokines/growth factors in the cellular milieu.

Animals↗
PubMed1994

Trypanosoma cruzi: effects of infection on receptor-mediated chronotropy and Ca2+ mobilization in rat cardiac myocytes.

Acute Trypanosoma cruzi infection is commonly associated with disorders of impulse conduction and muscle contraction in heart. In order to determine the extent to which receptor function changed in response to infection, infected neonatal rat cardiac myocytes in culture were compared with matched controls with regard to chronotropic response and Ca2+ mobilization following the application of adrenergic agonists. At 7-9 days in culture (5-7 days postinfection), spontaneous beat rates of control myocytes were four times as rapid as those in infected cells. Control cells responded to 10(-5) M isoproterenol (ISO) and 10(-6) M norepinephrine (NE) with increases in beat rate of 34 and 40%, respectively. Effects of ISO on infected cells were similar, and adenylate cyclase activity was similar in control and infected cells when measured in the presence of ISO alone or in combination with Gpp(NH)p. NE produced a more marked chronotropic response in infected cultures and altered Ca2+ mobilization. NE treatment increased Ca2+ levels in control cardiac myocytes from 51.8 +/- 4.4 to 113 +/- 16 nM (in 0 Ca2+ medium) and from 85.2 +/- 6.8 to 131.3 +/- 24.5 nM (1 mM external Ca2+). In infected cardiac myocytes, NE increased Ca2+ from 116.8 +/- 17 to 164.7 +/- 9.6 nM (in 0 Ca2+ medium) and from 132.2 +/- 13.2 to 162.5 +/- 0.3 nM (1 mM Ca2+ medium). Thus, basal and alpha-adrenergic-stimulated Ca2+ levels were higher in infected than uninfected myocytes regardless of the extracellular Ca2+ levels, although the fractional increase in infected myocytes was significantly lower than that in controls (1.4- and 1.2-fold vs 2.2- and 1.5-fold). Therefore, both chronotropic and Ca(2+)-mobilization responses to the alpha-adrenergic agonist NE are altered in T. cruzi-infected cardiac myocytes; the chronotropic response of similarly infected cells to the beta-adrenergic agonist ISO was not affected. These data indicating that T. cruzi infection may be associated with a dissociation in responses to these agonists suggest a possible mechanism to explain, in part, the cardiac dysfunction characteristic of Chagas' disease.

Animals↗
PubMed1994

Gap junction channels: distinct voltage-sensitive and -insensitive conductance states.

All mammalian gap junction channels are sensitive to the voltage difference imposed across the junctional membrane, and parameters of voltage sensitivity have been shown to vary according to the gap junction protein that is expressed. For connexin43, the major gap junction protein in the cardiovascular system, in the uterus, and between glial cells in brain, voltage clamp studies have shown that transjunctional voltages (Vj) exceeding +/- 50 mV reduce junctional conductance (gj). However, substantial gj remains at even very large Vj values; this residual voltage-insensitive conductance has been termed gmin. We have explored the mechanism underlying gmin using several cell types in which connexin43 is endogenously expressed as well as in communication-deficient hepatoma cells transfected with cDNA encoding human connexin43. For pairs of transfectants exhibiting series resistance-corrected maximal gj (gmax) values ranging from < 2 to > 90 nS, the ratio gmin/gmax was found to be relatively constant (about 0.4-0.5), indicating that the channels responsible for the voltage-sensitive and -insensitive components of gj are not independent. Single channel studies further revealed that different channel sizes comprise the voltage-sensitive and -insensitive components, and that the open times of the larger, more voltage-sensitive conductance events declined to values near zero at large voltages, despite the high gmin. We conclude that the voltage-insensitive component of gj is ascribable to a voltage-insensitive substate of connexin43 channels rather than to the presence of multiple types of channels in the junctional membrane. These studies thus demonstrate that for certain gap junction channels, closure in response to specific stimuli may be graded, rather than all-or-none.

Animals↗
PubMed1994

Conduction defects and arrhythmias in Chagas' disease: possible role of gap junctions and humoral mechanisms.

The protozoan parasite Trypanosoma cruzi causes Chagas' disease, a major cause of cardiac dysfunction in Latin Americans. Chagas' disease exhibits both acute and chronic phases, and each may be characterized by cardiac conduction disturbances. In acutely infected cultures of rodent heart cells, synchronized spontaneous beating becomes less regular, and coupling between cells is reduced. The basis of this decreased conduction is apparently in localization of the gap junction protein (Cx43) inside infected cells. Although total Cx43 is normal in infected cells, little is recognizable at appositional membranes. Electrophysiological properties are also altered by this infection. Action potentials are shortened, resting Ca2+ levels are elevated, and response to alpha-adrenergic agonists was altered, compared to controls. Humoral factors may contribute to the conduction defects in chronic Chagas' disease. Sera from chronically infected rabbits produced ECG abnormalities in Langendorff-perfused rabbit hearts. These findings indicate that chagasic infection may modify ion channel function in the heart, and we suggest that these changes may be manifested in the conduction disturbances that characterize this disease.

Action Potentials↗
PubMed1994

Norepinephrine induces Ca2+ release from intracellular stores in rat pinealocytes.

In rat pinealocytes, an increase in intracellular Ca2+ concentration ([Ca2+]i) due to Ca2+ influx in response to norepinephrine (NE) is a well recognized event involved in regulating several metabolic functions. Since NE also stimulates the metabolism of phosphatidyl inositols in rat pineal gland, it is conceivable that Ca2+ release from intracellular stores also contributes to the NE-induced increase in [Ca2+]i. In this communication, we report that in rat pinealocytes loaded with fura-2, a Ca2+ indicator, NE induced a transient increase in [Ca2+]i that preceded the known Ca2+ influx. This novel [Ca2+]i response to NE was detected in pinealocytes bathed with Ca2+-free saline and prevented by TMB-8, a blocker of Ca2+ release from intracellular stores, supporting the notion that the transient NE-induced Ca2+ response was due to Ca2+ release from intracellular stores. In addition, after an extended exposure to NE a new addition of this neurotransmitter did not elicit the phasic Ca2+ response, and application of increasing amounts of NE induced a Ca2+ response that was progressively smaller, suggesting desensitization. Thus, NE is proposed to increase [Ca2+]i in rat pinealocytes by two mechanisms: (1) phasic release from intracellular stores and (2) tonic influx through a mechanism activated by larger applications of NE than required to evoke the phasic release.

Animals↗
PubMed1994

Voltage-dependent gap junctional conductance in hepatopancreatic cells of Procambarus clarkii.

Properties of gap junction channels present between specific cell types constituting the hepatopancreas of the crayfish (Procambarus clarkii) were investigated using the dual whole cell voltage clamp technique. Four different cell types (E, Fe, R and B) were identified on the basis of their morphology using light and electron microscopy. Although junctional conductance (Gj) could not be measured in B-B cell pairs, junctional currents were resolved in both homologous and heterologous combinations of the other cell types. E-E, Fe-Fe, and E-Fe cell pairs exhibited strong dependence on inside-out voltage (Vi-o), such that Gj increased with hyperpolarization to a maximal plateau reached at approximately -40 mV and was abolished with depolarization > 10 mV. The Gj-Vi-o relationship can be described by a squared Boltzmann relation with A = 0.101 and V0 = 0.135 mV. In this system, sensitivity of the junctions to transjunctional voltage was slight, if present at all. Gating mechanisms were complex, as evidence by the presence of multiple unitary channel conductance states. Single channel recordings showed that large unitary conductances (> 200 pS) were generally found between E-E, Fe-Fe, and E-Fe cell pairs, whereas smaller channel sizes (< 90 pS) were detected between R-R cell pairs.

Animals↗
PubMed1994

Human connexin43 gap junction channels. Regulation of unitary conductances by phosphorylation.

Connexin43 is the major gap protein in the heart and cardiovascular system. Single channel recordings of human connexin43 gap junction channels exogenously expressed in transfected SKHep1 cells demonstrate two discrete classes of channel events, with unitary conductances of predominantly 60 to 70 and 90 to 100 pS when recorded with an internal solution containing CsCl as the major current-carrying ionic species and at moderate transjunctional voltages (< 60 mV). Human connexin43 expressed in SKHep1 cells displays multiple electrophoretic mobilities (apparent M(r), approximately 41 to 45 kD) when resolved in Western blots. Treatment of connexin43 from these cells with alkaline phosphatase collapses the bands into a single 41-kD species; application of alkaline phosphatase to the cell interior through patch pipettes yields channels that are predominantly of the larger unitary conductance. The smaller 60- to 70-pS unitary conductance values correspond to the most common channel size seen in cultured rat cardiac myocytes; these channels were more frequently observed after treatment with the phosphatase inhibitor okadaic acid, which was shown to increase phosphorylation of human connexin43 in these cells under similar conditions. Exposure to the protein kinase inhibitor staurosporine shifted the proportion of events toward the largest unitary conductance and resulted in decreased phosphorylation of human connexin43 in seryl residues in these cells. Thus, the unitary conductance of human connexin43 gap junction channels covaries with the phosphorylation state of the protein. This change in unitary conductance appears to be a unique effect of phosphorylation on gap junction channels, since it has not been observed for other ion channels that have thus far been evaluated.

Alkaloids↗
PubMed1993

Identification of proximal and distal regulatory elements of the rat connexin32 gene.

We have investigated the genetic basis of the transcriptional regulation of the rat connexin32 gene which encodes the major gap junction protein in rat liver. Primer extension analysis of RNA isolated from adult rat liver identified multiple initiation sites clustered between -110 bp and -50 bp upstream from the translation start codon. An approx. 760 bp genomic DNA fragment upstream of the first exon which included the mRNA start sites was cloned 5' to the luciferase reporter cassette in p19LUC to yield pCx32-800/-33-LUC. Transfection of pCx32-800/-33-LUC resulted in a 200-fold increase in luciferase activity above p19LUC in the human hepatoma cell line HuH-7. Using a series of vectors containing 5' deletions of the 760 bp fragment, a basal promoter was localized between -179 bp and -134 bp. Three DNA:protein complexes were identified with the basal promoter fragment by DNA mobility shift assay using nuclear extracts from HuH-7 cells. Two of the DNA-binding complexes appeared to be related to the transcription factor Sp1. In addition, three DNase hypersensitive (HS) sites were identified within the genomic locus of connexin32 in adult rat liver. Two of the DNase HS regions behaved as silencer elements with both the native promoter and a heterologous promoter in HuH-7 cells. These data demonstrate that (1) the active promoter responsible for rat connexin32 mRNA transcription is located upstream of the first exon, (2) a basal promoter region was localized to a 50 bp region which formed multiple DNA:protein complexes, and (3) multiple proximal and distant regulatory elements are involved in the expression of connexin32.

Animals↗
PubMed1993

Extracellular ATP4- modulates organic anion transport by rat hepatocytes.

The hepatocyte has an organic anion transport system that recognizes compounds such as bilirubin and sulfobromophthalein. These anions circulate bound tightly to albumin from which they are extracted rapidly by hepatocytes by an electroneutral process that requires extracellular inorganic anions such as Cl- for activity. Transport activity is reduced by depletion of intracellular ATP, but whether ATP interacts directly with this transporter is not known. In this study, the influence of extracellular ATP on the hepatocyte organic anion transport mechanism has been characterized. In the presence of 2.5 mM Ca2+ and 2 mM Mg2+, initial uptake of [35S]sulfobromophthalein was reduced by 50% at 1 mM ATP. In the absence of divalent cations sensitivity to ATP was 10-fold greater. Other nucleotides including UTP, CTP, GTP, ADP, AMP, and AMP-PCP (adenosine 5'-(beta,gamma-methylene)triphosphate) were inactive. Decreased transport activity was rapidly reversible, was non-competitive with respect to ATP, did not require ATP hydrolysis, and did not correlate with P2y purinergic receptor activity. Differential activity of ATP on sulfobromophthalein transport in the presence and absence of divalent cations was not due to ecto-ATPase activity but rather to alteration in [ATP4-]. Although an ATP4- receptor in macrophages mediates increased cellular permeability, reduced organic anion permeability is seen in hepatocytes. This effect is not seen in the hepatoma cell line HepG2. Modulation of activity of the organic anion transporter by extracellular ATP may have important pathophysiological consequences in conditions resulting in liver cell injury.

Adenosine Triphosphate↗
PubMed1993

Cytokine regulation of neuronal differentiation of hippocampal progenitor cells.

The signalling mechanisms governing haematolymphopoiesis and those regulating neural development may be closely related, as indicated by similarities of higher-order structure and function of the cytokines involved, of the regional and temporal regulation of their transcription and translation, and of their bioactivity. Here we investigate this possible evolutionary connection using retroviral transduction of a temperature-sensitive mutant form of the SV40 large T antigen to develop conditionally immortalized murine embryonic hippocampal progenitor cell lines. Treatment of these cells with cytokines that are thought to participate in progressive lymphoid maturation, immunoglobulin synthesis and erythropoiesis causes progressive neuronal differentiation, as defined by morphological criteria, successive expression of increasingly mature neurofilament protein, and the generation of inward currents and action potentials. The cytokine interleukin(IL)-11 induces expression of action potentials that are insensitive to tetrodotoxin, which is indicative of developmentally immature sodium channels. By contrast, for expression of more mature action potentials (tetrodotoxin-sensitive) one of the interleukins IL-5, IL-7 or IL-9 must be applied in association with transforming growth factor-alpha after pretreatment with basic fibroblast growth factor. Our results suggest that the mechanisms regulating lineage commitment and cellular differentiation in the neural and haematopoietic systems are similar. Further, they define an in vitro model system that may facilitate molecular analysis of graded stages of mammalian neuronal differentiation.

Action Potentials↗
PubMed1993

Evidence that myocardial pertussis toxin substrates are uniquely altered in acute murine Chagas' disease in a manner unrelated to myocardial dysfunction.

In an effort to correlate biochemical characteristics of the beta-adrenergic receptor complex with myocardial function, mouse myocardial GTP-binding proteins, specifically substrates for pertussis toxin (PT), were analysed with regard to the influence of infection with Trypanosoma cruzi, the causative agent of Chagas' cardiomyopathy. Infection was found to decrease in a non-uniform manner the magnitude of ADP-ribosylation in the PT substrates. High detergent concentrations attenuated the infection-associated decrease in PT-dependent ADP-ribosylation. Infection also altered the kinetics of the PT-dependent ADP-ribosylation reaction from a time course wherein maximal PT-dependent ADP-ribosylation occurred after 12 h incubation in control animals to one in which maximal PT-dependent ADP-ribosylation occurred after 3 h incubation and thereafter declined. Immunochemical analysis of the PT-substrates revealed an infection-associated decrease in alpha i1, alpha o, an increase in alpha i2 and no change in alpha i3. Verapamil treatment, which prevents the clinical consequences of infection, did not influence any of the infection-associated changes in PT-dependent ADP-ribosylation of GTP-binding protein substrates or their immunochemical properties. Complementary studies using isolated rat neonatal cardiocytes infected with the parasite further substantiated the finding that the infection-associated decrease in PT-dependent ADP-ribosylation and the associated change in the kinetics of the reaction were properties uniquely associated with the presence of the parasite.

Acute Disease↗
PubMed1993

Effect of tumor promoting stimuli on gap junction permeability and connexin43 expression in ARL18 rat liver cell line.

The ARL18 rat liver cell line has previously been used for screening tumor promoters in the metabolic cooperation assay (Williams 1980; Williams et al. 1981; Telang et al. 1982). These cells display high levels of gap junctional communication, as assessed functionally and immunologically. Intracellularly injected Lucifer Yellow diffused extensively and there was rapid fluorescent recovery after photobleaching. Moreover, expression of connexin43 (Cx43) was high as evaluated by immunocytochemistry of cell monolayers and Western blot analysis of total cell homogenates. Western blot analysis revealed multiple forms of Cx43, which presumably correspond to known dephosphorylated and phosphorylated states of this protein. Gap junction permeability and Cx43 expression in ARL18 cells were studied after exposure to the tumor promoters 12-0-tetradecanoyl-phorbol-13-acetate (TPA), and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane (DDT), and after wounding the cell monolayer. TPA and DDT strongly inhibited gap junction permeability; whereas monolayer wounding did not affect the degree of fluorescent recovery after injury, either in the cells on the edge of the wound or in distal regions. No changes in the cellular distribution of Cx43 were observed after any of these treatments, although Western blots revealed a decrease in total Cx43 after 24-h exposure to DDT (10 micrograms/ml) and a slight increase after TPA treatment (30 min, 0.1 microgram/ml). Relative abundance of different phosphorylated Cx43 forms was increased after 1 h exposure to DDT (10 micrograms) and 30 min exposure to TPA (0.1 microgram/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗