Judson D. Sheridan

Dye movement and low-resistance junctions between reaggregated embryonic cells

Abstract Reaggregated cells from Xenopus laevis embryos in blastula through larval stages become electrically coupled within 20 min of the beginning of aggregation. Some of these cells can exchange fluorescein injected intracellularly. The transfer of dye appears to depend on the presence of low-resistance junctions although the precise site of transfer is unknown. The relevance of these results to processes occurring in intact embryos is discussed.

Formation of low-resistance junctions in vitro in the absence of protein synthesis and ATP production☆

Abstract Formation of low-resistance junctions between Novikoff hepatoma cells was studied in the presence of inhibitors of protein synthesis or energy production. Cells were dissociated for 30 min in the presence of cycloheximide (CHX, 100 μg/ml), reaggregated by pelleting, and incubated for 60 min in the presence of CHX. This treatment (which inhibited protein synthesis by 99% over 30 min and prevented cell growth over 12 h) produced no significant difference in % of coupled cells or in coupling coefficient between control (0.346 ± 0.030) and treated cells (0.297 ± 0.034). Cells were treated for 12 h in CHX before dissociation and reaggregation with some decrease in % of coupled cells, but with no substantial reduction in coupling coefficient (0.298 ± 0.057). Multiple dissociations (and reaggregations) of the cells in the presence of CHX produced a decrease in 1 coupled cells and a slight reduction in coupling coefficients between treated (0.234 ± 0.029) and control preparations (0.312 ± 0.035). Cells were dissociated, treated with iodoacetate (IAA, 1 mM) for 30 min and reaggregated for 30 min. There was a small reduction in % coupled cells. Also the mean coupling coefficient was lower in treated cells (0.203 ± 0.041) compared to controls (0.393 ± 0.047), but this may reflect a general lowering of non-junctional membrane resistance. IAA resulted in a reduction of ATP to 5% of the control values. Thus, neither inhibition of protein synthesis nor severe reduction of ATP levels prevent or substantially reduce junction formation by Novikoff cells.

Metabolic interactions between animal cells through permeable intercellular junctions

Abstract In mixed cultures of HGPRT− (lacking hypoxanthine: guanine phosphoribosyltransferase activity) and wild-type mammalian cells coupled by permeable intercellular junctions, the purine nucleotide pools equilibrate between the two cell types prior to incorporation into nucleic acid. As a consequence, in the presence of exogenous hypoxanthine, the mutant cells stimulate the activity of HGPRT pathway in the wild-type cells and the de novo pathway of purine nucleotide biosynthesis is inhibited in both cell types. That is, the metabolic activity of a mixed culture of coupled cells is a unique characteristic of the mixture, not simply the sum of the activities of the component cells, whereas the activity of a mixed culture of cells which are not coupled by permeable intercellular junctions is simply the sum of the activities of the two cell types.

Cytochalasin B II: selective inhibition of cytokinesis in Xenopus laevis eggs.

Summary In the presence of cytochalasin B (CB), fertilized Xenopus laevis eggs fail to complete cleavage although nuclear division and furrowing occur normally. The effects of varied dose levels and changes in timing of exposure to CB indicate that a pool of substance(s) needed for subsequent cleavages becomes available for CB action just before first cleavage.

Gap junction assembly: roles for the formation plaque and regulation by the C-terminus of connexin43

Gap junction (GJ) “formation plaques” are distinct membrane domains with GJ precursors; they assemble by means of a series of defined steps. The C-terminus of Cx43 is required for normal progression of assembly, normal aggregation of 10-nm particles into small GJs, and negative regulation of assembly involving protein kinase C.

Cell Communication and Growth

The possibility that gap junctions play an important role in the control of cell proliferation (Furshpan and Potter, 1968; Loewenstein, 1968) has probably stimulated more interest and generated more controversy than any of their other suggested functions. Much of the attention given this idea has in fact been elicited by the corollary notion that defects in the junctions may contribute to the aberrant control of cell division in cancer (Loewenstein, 1968). Numerous comprehensive reviews, appearing in the last several years, have summarized the rather checkered history of these two related ideas (e.g., Loewenstein, 1979, 1981; Trosko et al., 1983; Sheridan and Atkinson, 1985; Vitkauskas and Canellakis, 1985). Rather than take a similar, and arguably redundant, approach, this chapter will focus on underlying concepts, using several more recent and selected research findings from various laboratories as illustrative examples.

Analysis of gap junctions and formation plaques between reaggregating Novikoff hepatoma cells

Freeze-fracture techniques have been used to study gap junction formation between Novikoff hepatoma cells reaggregated for 0–60 min. Quantitative data have been obtained for the proportion of membrane “interfaces” containing formation plaques with and without gap junctions [FP(+)s and FP(−)s, respectively], for the areas and numbers of FP(+)s and FP(−)s, and for the numbers and densities of 9- to 11-nm particles in FPs. Methods have been devised to correct these data for FP(+)s carried over from dissociation. Comparisons have also been made with undissociated Novikoff cells. The results show that new FP(−)s and FP(+)s first appear between 5 and 15 min of reaggregation, and increase gradually between 15 and 30 min. Interfaces with FP(−)s develop more rapidly than those with FP(+)s. Between 30 and 60 min, FP(−)s begin to disappear while FP(+)s continue to increase. The results suggest that many FP(−)s are converted into FP(+)s, but some FP(−)s may “abort.” The mean areas of FP(−)s and FP(+)s, either per plaque or per interface, show no significant changes with reaggregation time, although there is an increased average number of FPs per interface at later times. There is a progressive increase with time in the number of total 9- to 11-nm particles in FPs per plaque and per interface, with densities of 9- to 11-nm particles also generally increasing. However, FP(+)s, especially in 15-min samples, have a relatively constant density of unaggregated 9- to 11-nm particles, in spite of a large variation in aggregated particle density. A number of related analyses have led us to suggest that the 9- to 11-nm particles in FP(+)s behave as though they are in a saturated solution, with the aggregated particles being analogous to a precipitate. Particle aggregation in FP(−)s is considered analogous to precipitation from a supersaturated solution. The relation of the results to previous physiological data is discussed.

Decreased glucose stimulation threshold, enhanced insulin secretion, and increased beta cell coupling in islets of prolactin-treated rats

In order to determine the effect of lactogen on insulin secretion and junctional coupling among islet beta cells, ovine prolactin (oPRL) was infused by Alzet minipumps into female rats for 4 days. This treatment produced an oPRL level of 994 ± 122 ng/ml which, combined with residual rat PRL (rPRL) (12 ± 2 ng/ml), represented nearly a 20-fold increase from control (rPRL: 53 ± 17 ng/ml). In addition, plasma insulin was increased nearly 50% (control: 21.9 ± 3 μU/ml; experimental: 30.3 ± 3 μU/ml; p <0.05). When pancreata from lactogen-treated and control animals were perfused with linear 30–200 mg/dl glucose gradients, the apparent glucose threshold for insulin secretion in the experimental group was nearly 33% lower than that of the controls (i.e., 70 ± 4.6 mg/dl vs. 104 ± 7.5 mg/dl; p <0.01). The oPRL treatment also increased dye coupling among beta cells. Central cells in islets isolated from lactogen-treated and control animals were injected with Lucifer Yellow CH to estimate the extent of gap junctional coupling. There was nearly a twofold increase in the projected area of dye transfer per injection in the experimental vs. the controls: 4,607 ± 575 μm2 vs. 2,302 ± 474 μm2, respectively; p <0.02. The effects of oPRL decreased the apparent glucose threshold for insulin release, increased the above-threshold glucose-induced insulin secretion, and increased the extent of dye coupling among beta cells. These changes in insulin secretion and dye coupling closely resemble those observed in islets from pregnant rats.

Junctional transfer in cultured vascular endothelium: I. Electrical coupling

SummaryVascular endothelial cultures are composed of flat, polygonal monolayer cells which retain many of the growth, metabolic and physiological characteristics of the intimal endothelium. However, intercellular gap and tight junctions, which are thought to perform important roles in normal intimal physiology, are reduced in complexity and extent in culture. We have used electrophysiological techniques to test confluent (3- to 5-day) primary cultures of calf aortic (BAEC) and umbilical cord vein (BVEC) endothelium for junctional transfer of small ions. Both cell types are extensively electrically coupled. The passive electrical properties of the cultured cells were calculated from the decrease in induced membrane potential deflections with distance from an intracellular, hyperpolarizing electrode. Data analyses were based on a thin-sheet model for current flow (Bessel function). The generalized space constants (λ) were 208.6 μm (BAEC) and 288.9 μm (BVEC). The nonjunctional (6.14 and 8.72×108 Ω) and junctional (3.67 and 3.60×106 Ω) resistances were similar for the BAEC and BVEC, respectively. We detected no statistically significant differences in the resistance estimates for the two cell types.In vivo ultrastructural studies have suggested that aortic endothelium has more extensive gap junctions than venous endothelium. We have found that these ultrastructural differences are reduced in culture. The lack of any significant difference in electrical coupling capability suggests that cultured BAEC and BVEC have functionally similar junctional characteristics.

Modification of gap junctions in cells transformed by a temperature-sensitive mutant of Rous sarcoma virus

SummaryPrompted by our observation that a reduction in junctional permeance is one of the earlier events in the process of neoplastic transformation of a cell line by Rous sarcoma virus, we analyzed the gap junctions, from these cells to determine if the basis of the reduction is a loss of junctional channels. The cells (normal rat kidney, or NRK) are infected with a temperature-sensitive mutant of Rous sarcoma virus, allowing one easily to manipulate the cells into and out of the transformed state, and hence also to manipulate the junctional permeance. Using freeze-fracture electron microscopy, we found that the number and size of the junctions did not change in parallel with the permeance changes we had previously characterized. There is, however, a significant rearrangement of the junctional particles to a more random configuration when the cells are transformed and a reversal to the more ordered pattern when the cells are shifted back to the normal phenotype. These changes do parallel the changes in junctional permeance. We conclude that the permeance of existing junctional channels is modified and that the change in permeance may involve a change in the interaction of the junctional channels with each other and/or the surrounding lipid domain.