Ethanol Inhibits Gap-Junctional Coupling Between P19 Cells

Authors


  • Supported by the Canadian Institutes for Health Research and the Alcoholic Beverage Medical Research Foundation.

Reprint requests: Peter L. Carlen, Toronto Western Hospital, MCL-413, 399 Bathurst St., Toronto, ON, M5T 2S8, Canada; Email: carlen@uhres.utoronto.ca.

Abstract

Background: Gap junctions are plaques of multiple intercellular channels that connect the cytoplasm of adjacent cells. They provide both electrical and metabolic coupling and are an essential element in normal growth, development, and physiology. Little research exists on the relationship between alcohol administration and gap-junctional function or expression. This study looks at the function and expression of gap junctions after incubation and withdrawal of ethanol with P19 cell cultures.

Methods: Gap-junctional communication was assessed after 24 and 48 hr of exposure to 20 and 40 mM ethanol and after a 24-hr withdrawal period. The seeding technique was used, and diacyl-3,3′-indocarbocyanine iodide/calcein–stained donor cells were seeded on an unstained monolayer and then reviewed by confocal microscope and counted by flow cytometry. Analysis of connexin (Cx) proteins was performed by Western blot, gel electrophoresis, and immunoblots with antibodies for Cx26 and Cx43.

Results: All treatment regimens produced similar results, reducing dye coupling by more than 50% without recovery after a 24-hr withdrawal period. Exposing the cells to 20 mM ethanol for 48 hr did not significantly change the levels of Cx26 protein, but ethanol significantly decreased the levels of Cx43 in cultured P19 cells.

Conclusions: This study illustrates that ethanol can inhibit gap-junction function in the P19 cell line. Chronic exposure to 20 mM ethanol selectively decreased the levels of Cx43 protein in the membrane fraction of the cell cultures.

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