Identifying connexin expression and determining gap junction intercellular communication in rainbow trout cells

Abstract

Gap junctions are groups of membrane-bound channels that allow the passage of small molecules and ions between cells, permitting cell-cell communication. Because of their importance in cell homeostasis, gap junction presence and function were characterized in three commonly studied rainbow trout cell lines, namely RTgill-W1, RTgutGC, and RTG-2. Firstly, gap junction presence was determined by screening for gap junction protein alpha 7 and alpha 1 (GJA7 and GJA1) presence at the transcript level and GJA7 at the protein level. GJA7 was successfully identified at both the transcript and protein levels, and GJA1 was detected at the transcript level in all three cell lines. This is the first report of a GJA7 full-length transcript sequence in rainbow trout cells. Gap junction function, as determined by gap junction intercellular communication (GJIC), was examined using Lucifer yellow dye migration with the scrape and load technique; visualized by fluorescence microscopy. Phorbol 12-myristate 13-acetate (PMA), a gap junction inhibitor, was used to confirm the presence of functional gap junctions. Effects of serum deprivation on GJIC were also monitored; 24-h serum deprivation resulted in greater dye migration compared with 30-min serum deprivation. Both RTG-2 and RTgill-W1 showed significant dye migration that was inhibited by PMA while RTgutGC did not. Human foreskin fibroblast (HFF-1) cells were used as a positive control for gap junction presence and function. Taken together, our study shows that rainbow trout cells express connexin transcripts and proteins, and RTG-2 and, to a lesser extent, RTgill-W1 cells are able to perform GJIC.