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D C Spray

Publications and source records attributed to D C Spray.

At least 163 records · Page 9Linked to original sources
PubMed1987

Superoxide dismutase protects cultured neurons against death by starvation.

Brief substrate deprivation resulted in high mortality of superior cervical ganglion neurons in culture, assayed 2 hr later by trypan blue exclusion. Involvement of superoxide anions was indicated by several observations. Survival was increased significantly by prior treatment that induced cells to take up superoxide dismutase. During starvation, neurons reduced nitroblue tetrazolium to form the blue precipitate formazan, and the color change was blocked in neurons preloaded with superoxide dismutase. The incidence of staining was comparable to the mortality. In many cells, brief starvation caused the appearance of fluorescence due to oxidation of 2',7'-dichlorofluorescin to dichlorofluorescein, which indicates that oxidants were generated intracellularly. In some cells fluorescence was transient, as would be caused by membrane breakdown, and these cells were then shown to be dead. Superoxide generation caused by substrate deprivation may contribute importantly to cell damage in a variety of pathological conditions.

Animals↗
PubMed1987

Proteoglycans and glycosaminoglycans induce gap junction synthesis and function in primary liver cultures.

Intercellular communication via gap junctions, as measured by dye and electrical coupling, disappears within 12 h in primary rat hepatocytes cultured in serum-supplemented media or within 24 h in cells in a serum-free, hormonally defined medium (HDM) designed for hepatocytes. Glucagon and linoleic acid/BSA were the primary factors in the HDM responsible for the extended life span of the electrical coupling. After 24 h of culture, no hormone or growth factor tested could restore the expression of gap junctions. After 4-5 d of culture, the incidence of coupling was undetectable in a serum-supplemented medium and was only 4-5% in HDM alone. However, treatment with glycosaminoglycans or proteoglycans of 24-h cultures, having no detectable gap junction protein, resulted in synthesis of gap junction protein and of reexpression of electrical and dye coupling within 48 h. Most glycosaminoglycans were inactive (heparan sulfates, chondroitin-6 sulfates) or only weakly active (dermatan sulfates, chondroitin 4-sulfates, hyaluronates), the weakly active group increasing the incidence of coupling to 10-30% with the addition of 50-100 micrograms/ml of the factor. Treatment of the cells with 50-100 micrograms/ml of heparins derived from lung or intestine resulted in cells with intermediate levels of coupling (30-50%). By contrast, 10-20 micrograms/ml of chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, or liver-derived heparin resulted in dye coupling in 80-100% of the cells, with numerous cells showing dye spread from a single injected cell. Sulfated polysaccharides of glucose (dextran sulfates) or of galactose (carrageenans) were inactive or only weakly active except for lambda-carrageenan, which induced up to 70% coupling (albeit no multiple coupling in the cultures). The abundance of mRNA (Northern blots) encoding gap junction protein and the amounts of the 27-kD gap junction polypeptide (Western blots) correlated with the degree of electrical and dye coupling indicating that the active glycosaminoglycans and proteoglycans are inducing synthesis and expression of gap junctions. Thus, proteoglycans and glycosaminoglycans, especially those found in abundance in the extracellular matrix of liver cells, are important in the regulation of expression of gap junctions and, thereby, in the regulation of intercellular communication in the liver. The relative potencies of heparins from different tissue sources at inducing gap junction expression are suggestive of functional tissue specificity for these glycosaminoglycans.

Animals↗
PubMed1986

Gap junctional conductance and permeability are linearly related.

The permeability of gap junctions to tetraethylammonium ions was measured in isolated pairs of blastomeres from Rana pipiens L. and compared to the junctional conductance. In this system, the junctional conductance is voltage-dependent and decreases with moderate transjunctional voltage of either sign. The permeability to tetraethylammonium ions was determined by injecting one cell of a pair with tetraethylammonium and monitoring its changing concentration in the prejunctional and postjunctional cells with ion-selective electrodes. Junctional conductance was determined by current-clamp and voltage-clamp techniques. For different cell pairs in which the transjunctional voltage was small and the junctional conductance at its maximum value, the permeability to tetraethylammonium ions was proportional to the junctional conductance. In individual cell pairs, a reduction in the junctional conductance induced by voltage was accompanied by a proportional reduction in the permeability of the gap junction over a wide range. The diameter of the tetraethylammonium ion (8.0 to 8.5 A, unhydrated) is larger than that of the potassium ion (4.6 A, hydrated), the predominant current-carrying species. The proportionality between the permeability to tetraethylammonium ions and the junctional conductance, measured here with exceptionally fine time resolution, indicates that a common gap junctional pathway mediates both electrical and chemical fluxes between cells, and that closure of single gap junction channels by voltage is all or none.

Animals↗
PubMed1986

Sensitivity of gap junctional conductance to H ions in amphibian embryonic cells is independent of voltage sensitivity.

In vertebrate embryos gap junctional conductance (gj) is reduced by transjunctional voltage (Vj) and by cytoplasmic acidification; in each case sensitivity is comparable to those of other channels gated by voltage and ligand-receptor binding. We show here that the mechanisms by which Vj and intracellular pH (pHi) gate gj are apparently independent. Partial reduction of gj by lowering pHi neither attenuates nor enhances further reduction by Vj. Certain drugs irreversibly (glutaraldehyde, 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) or reversibly (retinoic acid) abolish dependence of gj on pHi without appreciably affecting kinetic properties of voltage dependence or the shape of the steady-state Vj-gj relation. These findings suggest that the mechanisms by which pHi and Vj act on the gap junction are at least partially distinct and presumably involve separate regions of the junctional macromolecules.

Animals↗
PubMed1986

Isolated liver gap junctions: gating of transjunctional currents is similar to that in intact pairs of rat hepatocytes.

We have shown previously that conductance of rat liver gap junctions is blocked by an affinity-purified polyclonal antibody generated against rat liver junctional membranes, is not affected by moderate transjunctional or transmembrane potentials, and is reversibly decreased by cytoplasmic acidification and perfusion with octanol. We have now recorded currents from isolated liver gap junctions using patch electrodes dipped through a layer of mixed lipids whose concentrations match those of isolated liver appositional membranes. These currents are blocked by the same polyclonal antibody, are insensitive to moderate voltages imposed across the pipette tip, and are reversibly blocked by similar concentrations of H ions and octanol as are junctions in situ. The currents are likely to be gap junctional in origin; their block by low pH and other agents indicates that the gating mechanisms are intrinsic to the gap junctions themselves and presumably result from conformational change in the channel-forming protein.

Animals↗
PubMed1986

cAMP increases junctional conductance and stimulates phosphorylation of the 27-kDa principal gap junction polypeptide.

Membrane-permeant cAMP derivatives (dibutyryl- and 8-bromo-cAMP) increase gap-junctional conductance within minutes when applied to voltage-clamped pairs of rat hepatocytes. Glucagon also increases junctional conductances, but the response has a more rapid onset and is more rapidly reversible. The glucagon effect can be prevented by intracellular injection of the protein inhibitor of the cAMP-dependent protein kinase (Walsh inhibitor), indicating that the catalytic subunit of cAMP-dependent protein kinase is directly involved. The 27-kDa major gap junction polypeptide is phosphorylated when liver cells dissociated into small groups are incubated with 32P. Addition of 8-bromo-cAMP to cells increases the incorporation of 32P into the 27-kDa junctional protein. Serine is the amino acid residue that is phosphorylated. When isolated liver gap junctions are incubated in the presence of catalytic subunit of the cAMP-dependent protein kinase, the 27-kDa gap junction polypeptide is phosphorylated with low stoichiometry on serine. The rapid increases in gap junctional conductance caused by agents that elevate cAMP and phosphorylation of the gap junction protein by cAMP-dependent protein kinase suggest that cAMP-dependent phosphorylation of the gap junction channel modulates the conductance of liver gap junctions.

Animals↗
PubMed1986

Electrophysiological properties of gap junctions between dissociated pairs of rat hepatocytes.

Physiological properties of isolated pairs of rat hepatocytes were examined within 5 h after dissociation. These cells become round when separated, but cell pairs still display membrane specializations. Most notably, canaliculi are often present at appositional membranes which are flanked by abundant gap and tight junctions. These cell pairs are strongly dye-coupled; Lucifer Yellow CH injected into one cell rapidly diffuses to the other. Pairs of hepatocytes are closely coupled electrically. Conductance of the junctional membrane is not voltage sensitive: voltage clamp studies demonstrate that gj is constant in response to long (5 s) transjunctional voltage steps of either polarity (to greater than +/- 40 mV from rest). Junctional conductance (gj) between hepatocyte pairs is reduced by exposure to octanol (0.1 mM) and by intracellular acidification. Normal intracellular pH (pHi), measured with a liquid ion exchange microelectrode, was generally 7.1-7.4, and superfusion with saline equilibrated with 100% CO2 reduced pHi to 6.0-6.5. In the pHi range 7.5-6.6, gj was constant. Below pH 6.6, gj steeply decreased and at 6.1 coupling was undetectable. pHi recovered when cells were rinsed with normal saline; in most cases gj recovered in parallel so that gj values were similar for pHs obtained during acidification or recovery. The low apparent pK and very steep pHi-gj relation of the liver gap junction contrast with higher pKs and more gradually rising curves in other tissues. If H+ ions act directly on the junctional molecules, the channels that are presumably homologous in different tissues must differ with respect to reactive sites or their environment.

Animals↗
PubMed1986

Cutaneous temperature receptors.

Specific thermoreceptors comprise an electrophysiologically distinct class of cutaneous receptors with a morphological substrate (free nerve endings) and plausible transduction mechanism (electrogenic Na pump with or without auxiliary temperature-dependent processes). Because responses to thermal and mechanical stimuli converge along the neural pathway, we have difficulty explaining the purity of cold and warm sensations; participation of dual-modality receptors in sensory discrimination cannot be ruled out. The field is now at a point where a leap in understanding would be achieved by intracellular recordings from the sensory receptor (for which patch clamp studies on isolated neuronal elements may provide the necessary technology) and from continued analysis of what information is lost and what retained in passage from one synapse to the next along the thermal pathway.

Animals↗
PubMed1986

Effects of protein reagents on electrotonic coupling in crayfish septate axon.

The lateral giant axons of the crayfish nerve cord are composed of segments contributed by each ganglion, which are electrotonically coupled by way of gap junctions. We have investigated the involvement of protein residues in regulating the resistance of crayfish junctional channels by determining effects of group-specific protein reagents. When applied to well-coupled axons, the sulfhydryl group reagents N-ethylmaleimide (NEM) and diamide uncoupled the segments; junctional resistance (Rj) was increased without changing membrane resistance or axoplasmic pH (pHi). The uncoupling produced by NEM could be reversed by alkalinization of the cytoplasm (addition of ammonium chloride to the external medium). Another sulfhydryl reagent (p-chloromercuribenzoic acid) increased Rj to a lesser extent. A disulfide reagent and three amino and three carboxyl group reagents had no effect on the Rj of these axons. The effect of group-specific reagents on partially uncoupled axons was tested by applying the drugs to axons previously exposed to weak acids. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline recoupled partially uncoupled axons by decreasing Rj and prevented subsequent uncoupling of the junction by low pHi. Another carboxyl group reagent, as well as sulfhydryl and amino group reagents, either had no effect or uncoupled the axons further by increasing Rj. These experimental results suggest that amino acid residues, possibly containing carboxyl and sulfhydryl groups, control the opening and closing of junctional channels and may thus be associated with the channels' active sites.

Amino Acids↗
PubMed1986

Effect of calcium on the dissociation of the mature rat heart into individual and paired myocytes: electrical properties of cell pairs.

The dissociation of adult rat heart into individual, functionally intact, calcium-tolerant myocytes requires precise manipulation of extracellular calcium levels. Dissociation of intercellular connections is achieved by lowering extracellular calcium to micromolar levels for a short period. By imposing a very small increment in free calcium activity (from 14 to 17 microM) during this period, we achieve a significant yield of functionally intact pairs of myocytes still joined at the intercalated disc. We obtain fewer intact cells, but many of these are paired end to end. These findings permit us to describe some structural characteristics of intercellular connections between cardiac cells and to report unambiguous measurements of electrotonic coupling and dye transfer between rat cardiac cell pairs. We find that the strength of electrical coupling between cells isolated as pairs with intact junctional contacts is much greater than that measured between cell pairs that have formed new junctional contacts.

Animals↗
PubMed1986

Extracellular matrix regulation of cell-cell communication and tissue-specific gene expression in primary liver cultures.

Epithelial-mesenchymal interactions are effected, in part, by extracellular matrix components. We have spent many years analyzing the influence of extracellular matrix, both as extracts of matrix and as purified matrix components, on the growth and differentiation of normal and neoplastic liver cells. Currently we are focused on analyzing the influence of the extracellular matrix components, glycosaminoglycans and proteoglycans. We have found that these factors induce dramatic morphological changes, are potent inducers of gap junction synthesis and can regulate tissue-specific gene expression. With respect to gap junctions: intercellular communication via gap junctions, as measured by dye and electrical coupling, disappears within 12 hrs in primary rat hepatocytes cultured in serum supplemented media or within 24 hrs in cells in a serum free, hormonally defined medium designed for hepatocytes. Glucagon and linoleic acid/BSA were the primary factors in the HDM responsible for the extended life span of the electrical coupling. Addition of proteoglycans or glycosaminoglycans to hormonally defined medium after 24 hrs resulted in reexpression of electrical and dye coupling when assayed at 96 hrs of culture. The incidence of coupling was less than 5% in hormonally defined medium alone. Coupling incidence increased to 10-30% with the addition of 10 micrograms/ml of glycosaminoglycans (i.e., hyaluronic acid, dermatan sulfate, chondroitin 4- or 6-sulfate, and iota- or kappa-carrageenan) to hormonally defined medium. By contrast, the same concentrations of chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, or lambda-carrageenan resulted in dye coupling in more than 70% of the cells, with numerous cells showing dye spread from a single injected cell (in the case of the proteoglycans). The greatest effect of those tested was elicited by the dermatan sulfate proteoglycans, which induced cell-cell communication in 90-100% of the cells. Heparins gave intermediate responses (30-50%). Western blots demonstrated that the amounts of the main intrinsic gap junction polypeptide (27 KDa) extractable from cells correlated with the degree of electrical and dye coupling. Thus, proteoglycans and glycosaminoglycans appear to elicit the formation and function of gap junctions and may thus play a role in the regulation of intercellular communication under normal and pathological conditions. With respect to gene expression: normal rat hepatocytes maintained in culture on tissue culture plastic and in serum supplemented medium lose their tissue-specific functions within hours to a few days due to loss of synthesis and to rapid degradation of tissue-specific mRNAs.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Cell junctions in early embryos of squid (Loligo pealei).

Squid embryos examined by freeze-fracture and thin-section electron microscopy exhibit identifiable gap junctions during mid-cleavage stages (stages 7-8), and junctional complexes composed of adherent appositions, elaborate septate junctions and gap junctions at slightly later stages (stages 12-13). During germinal layer establishment (stages 12-13) cytoplasmic bridges frequently link the embryonic cells. The presence of gap junctions in cleavage-stage embryos provides the morphological substrate for a demonstrated pathway of direct cell-cell communication that is modifiable by experimental treatments and may be physiologically regulatable. The existence of septate junctions and adherent contacts at later stages suggests that some functional specialization, perhaps the establishment of a strongly joined framework of cells at the surface of the embryo, accompanies the formation of germinal layers.

Animals↗
PubMed1985

Reduction of gap junctional conductance by microinjection of antibodies against the 27-kDa liver gap junction polypeptide.

Antibody raised against isolated rat liver gap junctions was microinjected into coupled cells in culture to assess its influence on gap junctional conductance. A rapid inhibition of fluorescent dye transfer and electrical coupling was produced in pairs of freshly dissociated adult rat hepatocytes and myocardial cells as well as in pairs of superior cervical ganglion neurons from neonatal rats cultured under conditions in which electrotonic synapses form. The antibodies have been shown by indirect immunofluorescence to bind to punctate regions of the plasma membrane in liver. By immunoreplica analysis of rat liver homogenates, plasma membranes, and isolated gap junctions resolved on NaDodSO4/polyacrylamide gels, binding was shown to be specific for the 27-kDa major polypeptide of gap junctions. This and similar antibodies should provide a tool for further investigation of the role of cell-cell communication mediated by gap junctions and indicate that immunologically similar polypeptides comprise gap junctions in adult mammalian cells derived from all three germ layers.

Animals↗
PubMed1985

Some electrical and pharmacological properties of gap junctions between adult ventricular myocytes.

Ventricular myocytes were isolated from adult rat hearts using the technique of Wittenberg and Robinson (Cell Tissue Res. 216: 231-251, 1981). These cells exhibited morphology, input resistance, time constant, and excitability expected for cells in intact cardiac tissue. Pairs of these cells were electronically coupled, and junctional conductance was unaffected by transjunctional potential or hyperpolarization of both cells. Brief exposure of cell pairs to medium equilibrated with 100% CO2 or containing 0.1 mM octanol quickly and reversibly decreased junctional conductance. We conclude that gap junctions between pairs of ventricular myocytes possess physiological properties like those of junctions in many other tissues. This preparation will be useful in evaluating drug action on junctional communication in heart.

Animals↗
PubMed1985

Regulation of gap junctional conductance.

Gap junctional conductance is regulated by the number of channels between coupled cells (the balance between formation and loss of these channels) and by the fraction of these channels that are open (gating mechanisms). A variety of treatments are known to affect junction formation. Adenosine 3',5'-cyclic monophosphate (cAMP) is involved in some cases, and protein synthesis may be required but precursor molecules can also exist. Junction removal occurs both by dispersion of particles and by internalization of junctional membrane. Factors promoting removal are not well understood. A variety of gating mechanisms exist. Coupling may be controlled by changes in conductance of nonjunctional membranes. Several kinds of voltage dependence of junctional conductance are known, but rat ventricular junctions at least are electrically linear. Cytoplasmic acidification decreases conductance of most gap junctions. Sensitivity in rat ventricular myocytes allows modulation of coupling by moderate changes near normal internal pH. Increasing intracellular Ca also decreases junctional conductance, but in the better studied cases sensitivity is much lower to Ca than H. A few data support low sensitivity to Ca in cardiac cells, but quantitative studies are lacking. Higher alcohols such as octanol block junctional conductance in a wide range of tissues including rat ventricular myocytes. An antibody to liver gap junctions blocks junctions between rat ventricular myocytes. Cross reactivity indicates at least partial homology between many gap junctions. Although differences among gap junctions are known, a general physiology is being developed, which may have considerable relevance to normal cardiac function and also to conduction disorders of that tissue.

Animals↗
PubMed1985

Fate of intercellular junctions in isolated adult rat cardiac cells.

Freeze fracture and thin section techniques have revealed morphological changes in gap junctions and intercalated discs of adult rat myocytes following enzymatic dissociation. Cell separation leaves behind small vesicular remnants of formerly adjacent cells connected to the intact cell by gap junctions; in contrast, desmosomes cleave at the region of intercellular contact. Apparently, the next step in gap junction breakdown is internalization of the remnants. In thin section, lanthanum penetration reveals that the cleft of some apparently internalized gap junctions is in contact with the sarcolemma, while that of others is not. In freeze fracture replicas, cytoplasmic gap junctions frequently possess hexagonally packed domains of E-face pits separated by smooth regions that may correspond to separations of membranes of internalized junctions found in thin section. Study of material maintained overnight at 37 degrees C showed no surface junctional remnants; topologies of cytoplasmic gap junctions were generally complex, and concentric membrane vesicles were common. These observations suggest that enzymatic dissociation initiates a progressive, defined sequence of junctional internalization that begins with attached cell remnants and may have as the last determinable step the separation into single membranes inside the intact cell.

Animals↗
PubMed1984

pH dependence of transmission at electrotonic synapses of the crayfish septate axon.

Gap junctions between segments of the crayfish septate axon mediate electrotonic transmission of impulses propagating along the length of the nerve cord. We simultaneously measured intracellular pH (pHi) and gap junctional conductance (gj) while axons were exposed to saline equilibrated with CO2, weak acids, and the weak base ammonium chloride. Normal pHi is about 7.1. When pHi is elevated, gj is unaffected. When pHi is reduced, gj declines with an apparent pK of about 6.7 and a Hill coefficient of about 2.7. We also measured effects of pHi on non-junctional conductance (gnj) and on the coupling coefficient, k. Over the pHi range 6.2-8, gnj increases approximately linearly with pHi. Since k is a function of gj and gnj, it reached a maximum at about pHi 7.1, decreasing at higher and lower pHi. The pHi dependence of gj in crayfish septate axon is less steep and has a lower apparent pK than the gj-pHi relation in two vertebrate embryos previously examined. This finding illustrates a difference in gating among analogous and possibly homologous membrane channels.

Animals↗