Development and characterization of a dye-coupling assay to study gap junctional communication in smooth muscle cells.

A simple dye-coupling assay was developed to study gap junctional communication in cultured rat uterine smooth muscle cells, as well as in cultures from an established cell line derived from a human uterine leiomyosarcoma (SK-UT-1). In this system, the establishment of gap junctional communication is largely independent of cellular energy supplies, and synthesis of new protein is not a necessary prerequisite. Perturbing intracellular pH by treatment with acetic acid, propionic acid, ammonium chloride or ortho-nitrobenzyl acetate reduced dye-coupling only at cytotoxic concentrations. Increasing intracellular calcium concentrations by treatment with A23187 or ouabain significantly reduced dye-coupling, but was associated with dramatic alterations of cell morphology. Treatment of cultures with isoproterenol/theophylline or dibutyryl-cAMP significantly increased dye-coupling. At least one component of this increase was dependent on active translation but did not require active transcription. Both nocodazole and colchicine altered dye-coupling at high concentrations, but were ineffective at concentrations routinely used to alter microtubule function. Disruption of microfilaments with cytochalasin-D significantly reduced dye-coupling, but was associated with severely altered morphology. Periodate oxidation of terminal sialic acid residues on the dye-recipients did not affect dye-coupling, while removal of these residues with neuraminidase increased dye-coupling. Treatment of dye-recipients with tunicamycin did not significantly alter dye-coupling at concentrations that reduced cell replication. Wheat germ agglutinin, soy bean agglutinin and Ulex europaeus-I significantly reduced dye-coupling in a concentration dependent manner. Ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether and DDT all significantly reduced dye-coupling with an apparent concentration dependency. The results suggest that under the conditions used in this assay, gap junctions are relatively insensitive to alterations of intracellular pH and calcium ion concentrations. The results also suggest that this assay may prove useful for the identification of inhibitors of gap junctional communication. The data support the hypothesis that, in this system, the establishment of gap junctional communication is dependent on cell-cell recognition events that precede gap junctional assembly.Ph.D.BiologyPublic healthUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/162271/1/8920645.pd