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

Epstein, Miles L.; Sheridan, Judson D.; Johnson, Ross G.

doi:10.1016/0014-4827(77)90064-7

Cited by 75 publications

(20 citation statements)

References 19 publications

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“…(Compare with work on other cell cultures: Epstein, Sheridan & Johnson, 1977;Williams & DeHaan, 1981.) The leftovers were all of relatively large size; the sizes with 10-20 particles, abundant before, were missing (Table 7).…”

Section: Gap-junctional Membrane Particlesmentioning

confidence: 90%

Cell junction and cyclic AMP: I. Upregulation of junctional membrane permeability and junctional membrane particles by administration of cyclic nucleotide or phosphodiesterase inhibitor

1981

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Mammalian cells in culture were exposed to cyclic AMP, dibutyryl cyclic AMP, the phosphodiesterase inhibitor caffeine, or a combination of the last two, while junctional molecular transfer was probed with the series of microinjected, fluorescent-labelled linear molecules Glu, Glu-Glu, Glu-Glu-Glu, and Leu-Leu-Leu-Glu-Glu. The junctional permeability for these molecules increased with each of the agents, most markedly with the dibutyryl cyclic AMP-caffeine combination, as the intracellular cyclic nucleotide concentration rose. The junctional permeability effect developed over several hours. When probed with molecules close to the limit of cell-to-cell channel permeation (the most sensitive setting), the effect was detectable both, as an increase in the (relative) junctional transit rate and as an increase in the number of transferring cell interfaces in the test populations. The number of transferring cell interfaces reached a maximum by 4 hr, when the junctional transit rate, hence the junctional permeability, was still rising. Nonjunctional membrane permeability for the probe molecules, as determined by intracellular fluorescence loss, was not significantly changed (nor was there significant nonjunctional cell-to-cell transfer of molecules before or after the treatments). The rise in junctional permeability was associated with an increase in the number of gap junctional membrane particles, as determined by freeze-fracture electron microscopy: the average size of the particle clusters increased, and the frequency of the clusters increased, particularly that of the smaller (and presumably newer) clusters. This effect was blocked by treatments with the protein synthesis inhibitors cycloheximide or puromycin. These agents caused particle diminution (diminution of cluster frequency but not of average cluster size), with or without cyclic nucleotide. The junctional effects may represent a cyclic AMP-promoted proliferation of cell-to-cell channels. Some physiological implications, in particular, implications for hormone-regulated tissues, are discussed.

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Section: Gap-junctional Membrane Particlesmentioning

confidence: 90%

Cell junction and cyclic AMP: I. Upregulation of junctional membrane permeability and junctional membrane particles by administration of cyclic nucleotide or phosphodiesterase inhibitor

1981

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“…For example, Flagg-Newton et al (55) have reported that the number of gap junctional plaques detectable by freeze-fracture electron microscopy in Balb-3T3 fibroblasts after a 24-h treatment with 10 g/ml CHX was ϳ25% of that in untreated controls, which would correspond to a t1 ⁄2 of 12 h. Moreover, the gap junctions remaining were exceptionally large, leading to a less than 40% reduction in the gap junction area/m 2 of total cell surface. Similarly, Epstein et al (56) demonstrated that the capacity of Novikof hepatoma cells to assemble endogenously expressed Cx43 into functional gap junctions in a dissociation-reaggregation assay was only minimally affected by a 12-h pretreatment with 100 g/ml CHX. Our demonstration that protein synthesis inhibitors decrease the rate of connexin degradation provides an explanation for these previously puzzling observations.…”

Section: Discussionmentioning

confidence: 99%

Regulation of Connexin Degradation as a Mechanism to Increase Gap Junction Assembly and Function

Musil

VanSlyke

et al. 2000

Journal of Biological Chemistry

203

151

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Connexins, the integral membrane protein constituents of gap junctions, are degraded at a rate (t1 ⁄2 ‫؍‬ 1.5-5 h) much faster than most other cell surface proteins. Although the turnover of connexins has been shown to be sensitive to inhibitors of either the lysosome or of the proteasome, how connexins are targeted for degradation and whether this process can be regulated to affect intercellular communication is unknown. We show here that reducing connexin degradation with inhibitors of the proteasome (but not with lysosomal blockers) is associated with a striking increase in gap junction assembly and intercellular dye transfer in cells inefficient in both processes under basal conditions. The effect of proteasome inhibitors on wild-type connexin stability, assembly, and function was mimicked by treatment of assembly-inefficient cells with inhibitors of protein synthesis such as cycloheximide. Sensitivity of connexin degradation to cycloheximide, but not to proteasome inhibitors, was abolished when connexins were rendered structurally abnormal by perturbation of essential disulfide bonds or by mutation. Our findings provide the first evidence that intercellular communication can be up-regulated at the level of connexin turnover and that a short-lived protein may be required for conformationally mature connexins to become substrates of proteasomal degradation.

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“…We have not determined the ATP levels in our cells. M. Epstein and J. Sheridan (personal communication) found an ATP remnant of 20% in Novikoff hepatoma cell cultures with established junctions after a !-hr exposure to 1 mM iodoacetate, with persistent electrical coupling (see also Epstein, Sheridan & Johnson, 1977, for action of iodoacetate on forming junction).…”

Section: On Resistance To Junctional Uncouplingmentioning

confidence: 98%

Experimental depression of junctional membrane permeability in mammalian cell culture. A study with tracer molecules in the 300 to 800 dalton range

Flagg-Newton

Loewenstein

1979

J. Membrain Biol.

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Cell-to-cell junctional permeability in mammalian cell cultures was probed with a series of fluorescent tracers ranging 300 to 800 in molecular weight, during treatment with metabolic inhibitors, Ca-transporting ionophore, and carbon dioxide. Treatment with the combination of cyanide and iodoacetic acid (1--2 mM each), but not with either one alone, caused reversible junctional blockade to all tracer molecular species, large and small. (Electrical coupling, however, persisted in a proportion of the junctions tested.) Treatment with the ionophore A23187 (2--10 micrometers) or with CO2 (an atmosphere of 100% CO2 equilibrated with the medium) produced selective junctional blockade: transmission of a 688 and an 817-dalton tracer was generally blocked, while that of a 376-dalton traced and, in certain conditions, that of a 559-dalton one, persisted. The junctional effect of the ionophore required the presence of Ca in the external medium; and effective junctional blockade by CO2 required pretreatment in medium with high Ca concentration or, interchangeably, pretreatment in medium with high CO2 concentration. In one cell type, prolonged exposure to medium with high Ca concentration alone sufficed to block transmission of the 688-dalton tracer. These effects are discussed in terms of the Ca hypothesis of junctional permeability regulation. In comparison with mammalian (or other vertebrate and invertebrate) organized tissues or with insect cell cultures, the mammalian cell cultures are more resistant to junctional blockade. This difference in transmission stability is discussed in terms of the electron-microscopic finding in the mammalian cultures of fine, bilateral cell processes connected by gap junctions.

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

Cited by 75 publications

References 19 publications

Cell junction and cyclic AMP: I. Upregulation of junctional membrane permeability and junctional membrane particles by administration of cyclic nucleotide or phosphodiesterase inhibitor

Cell junction and cyclic AMP: I. Upregulation of junctional membrane permeability and junctional membrane particles by administration of cyclic nucleotide or phosphodiesterase inhibitor

Regulation of Connexin Degradation as a Mechanism to Increase Gap Junction Assembly and Function

Experimental depression of junctional membrane permeability in mammalian cell culture. A study with tracer molecules in the 300 to 800 dalton range

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