sq-23377 and lucifer-yellow

To investigate in bovine and embryonic chicken lens cultures the effects of elevated intracellular calcium on the permeability of gap junctions. To determine the changes in intracellular calcium using fura-2. To detect any changes in the phosphorylation of connexin43 after ionophore treatment.. Lucifer yellow was micro-injected into individual cells, and dye spread to neighboring cells was evaluated. Intracellular calcium levels were measured using the calcium indicator, fura-2. Cultures were also labeled with 32P-orthophosphate followed by immunoprecipitation with antibodies specific for the gap junction protein, connexin43.. Bovine lens cultures incubated in the presence of either A23187 or ionomycin showed a reduction in intercellular dye transfer. The intracellular calcium concentrations in bovine cells were increased from a mean value of 0.11 +/- .009 microM in the controls to a mean of 0.40 +/- .073 microM with ionomycin treatment. Subsequent addition of EGTA to the medium decreased the intracellular calcium concentrations to a mean of 0.26 +/- .113 microM and reversed the inhibition of dye spread found with ionomycin. With ionomycin in the medium, the phosphorylated form of connexin43 was not as prominent as in the controls. In contrast, these same treatments had no detectable effect on junctional permeability in the embryonic chicken lens cultures. Dye spread was equally extensive and rapid under control and ionophore conditions, even though fura studies showed an elevation in intracellular calcium levels.. In the bovine cultures, physiologically relevant changes in the levels of cytoplasmic calcium markedly reduced dye transfer. The increase in cytoplasmic calcium was correlated with a change in the phosphorylation level of connexin43. The regulation of junctional communication in the chick lens cultures appears to differ significantly from that in the bovine system.

Topics: Animals; Calcimycin; Calcium; Cattle; Cell Communication; Cell Membrane Permeability; Cells, Cultured; Chick Embryo; Connexin 43; Fura-2; Gap Junctions; Ionomycin; Isoquinolines; Lens, Crystalline; Phosphorylation

To investigate communication competence of cultured rat mesangial cells, Lucifer yellow transfer was studied using microinjection and scrape-loading techniques. Both methods yielded results indicating considerable gap junctional communication between cultured mesangial cells. Gap junctional communication between mesangial cells was upregulated by adenosine 3',5'-cyclic monophosphate (cAMP). Conversely, cell-to-cell communication was attenuated by exposure to the tumor promoter phorbol myristate acetate, the Ca ionophore ionomycin, reduced oxygen intermediates, and cell acidification. Expression of voltage gated calcium channels by mesangial cells was studied microspectrofluorimetrically using fura-2 fluorescence. KCl-induced depolarization, BAY-K 8644, and readdition of calcium to Ca-free depolarizing medium all produced a nifedipine-inhibitable increase in cytosolic calcium concentration. The existence of voltage-gated calcium channels in communication-competent cells suggests the possibility of propagation of depolarizing signals across the syncytium. This was studied by microapplication of KCl to the microenvironment of a single cell and monitoring fura-2 fluorescence in remote cells. This maneuver resulted in propagating calcium waves in communication-competent monolayers; calcium waves could not be evoked in monolayers exposed to an alkanol-type gap junction uncoupler, octanol. It is concluded that cultured rat mesangial cells form a syncytium capable of propagating calcium transients from a single depolarized cell to its coupled neighbors.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Biological Transport; Calcium; Calcium Channels; Cell Communication; Cell Division; Cells, Cultured; Fura-2; Giant Cells; Glomerular Mesangium; Intercellular Junctions; Ionomycin; Isoquinolines; Microinjections; Microscopy, Fluorescence; Octanols; Rats; Tetradecanoylphorbol Acetate