Changes in cytosolic CA2+ are not involved in DDT-induced loss of gap junctional communication in WB-F344 cells

Recent studies have demonstrated that the insecticide DDT is a tumor promoting agent. Similar to many other tumor promoting agents, DDT has been shown to inhibit gap junctional intercellular communication (GJIC) between cells in culture, and it has been suggested that DDT-induced loss of communication between adjacent cells may depend on changes in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). In the present study, the role of[Ca²⁺]_i in DDT-induced loss of GJIC was investigated in WB-F344 rat liver cells using the scrape-loading/dye transfer assay (SLDT) and the Ca²⁺ fourescent indicator, furà-2. Our results show that DDT at non-cytotoxic concentrations caused a reversible loss of GJIC. Inhibition of GJIC was not associated with detectable increases in [Ca²⁺]_i, and was not prevented by loading cells with the intracellular Ca²⁺ chelator, BAPTA. In addition, the hydroquinone, tBuBHQ, which caused a 2+3 fold sustained increase in [Ca²⁺]_i, did not inhibit GJIC. Conversely, when untreated cells were loaded with increasing BAPTA concentrations, GJIC were lost. These results indicate that increases in [Ca²⁺]_i are not responsible for DDT-induced loss of communication and that, in general an increase in [Ca²⁺]_i, within physiological levels is not sufficient to abolish GJIC. However, Ca²⁺-dependent processes that are active at normal resting [Ca²⁺_i appear to be required for the maintenance of GJIC.Abbreviations [Ca²⁺] cytosolic free Ca²⁺ concentration - GJIC gap junctional intercellular communication - SLDT scrape-loading/dye transfer assay - DDT 1,1,1-trichloro-2,2-di-(4-chlorophenyl)ethane - tBuBHQ 2,5-di(tert-butyl)-1,4-benzohydroquinone - LDH lactate dehydrogenase - ER endoplasmic reticulum - Fura-2 1-[2-(5carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxyl]-2-(2amino-5-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid - BAPTA bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraaceticacid - Fura-2/AM and BAPTA/AM are the cell permeant acetoxymethyl ester forms of fura-2 and BAPTA, respectively