Upregulation of gap junctional intercellular communication in immortalized gonadotropin-releasing hormone neurons by stimulation of the cyclic AMP pathway.
Increased gap junctional intercellular communication induced by agents that stimulate the adenylyl cyclase/cAMP pathway was observed in the GnRH-secreting neuronal cell line, GT1-7, and possible underlying mechanisms were examined. A 24-hour treatment of GT1-7 neurons with 100 microM dibutyryl cAMP + 100 microM IBMX or with 2 microM forskolin increased by greater than 2-fold the percentage of cells that were dye coupled, using the noninvasive dye coupling assay, fluorescent recovery after photobleaching (FRAP). Longer treatment times (48 h) and higher concentrations of dibutyryl cAMP (500 microM) did not further increase the percentage of dye-coupled cells, while there was no increase in dye coupling observed between untreated cells and cells treated for 2 h or less. The increase in dye coupling induced by dibutyryl cAMP/IBMX was inhibited by octanol or dieldrin, agents known to block gap junction-mediated intercellular coupling in other cell types. Western blot analysis of total protein or membrane protein-enriched extracts revealed no apparent difference in the cellular levels of connexin 26, a connexin subtype previously shown to be expressed by GT1-7 cells, between untreated cells and cells treated for 24 h with dibutyryl cAMP/IBMX or forskolin. In addition, expression of connexin 32 or 43 protein before or after treatment was not detected. On the other hand, a dramatic increase in both the number of neurites and neurites that immunostained positive for connexin 26 was observed in dibutyryl cAMP/IBMX-treated cells. We hypothesize that the observed increase in dye coupling between GT1-7 neurons following stimulation of the adenylyl cyclase/cAMP pathway results from an augmentation of cell-cell contacts due to an increased number of neurites containing gap junctional plaques, possibly through an effect on cellular differentiation.
Department of Pediatrics and Human Development, Michigan State University, East Lansing 48824, USA.
D F
DF
Matesic
T
T
Hayashi
J E
JE
Trosko
J A
JA
Germak
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002450:Cell Communication; D002455:Cell Division / Q000187:drug effects; D000072259:Connexin 26; D017630:Connexins / Q000378:metabolism; D000242:Cyclic AMP / Q000819:agonists / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D017629:Gap Junctions / Q000502:physiology*; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000502:physiology*; D013194:Staining and Labeling; D013268:Stimulation, Chemical; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000473:pathology
D017630:Connexins; D005456:Fluorescent Dyes; C000607043:Gjb2 protein, mouse; D000072259:Connexin 26; D007987:Gonadotropin-Releasing Hormone; D003994:Bucladesine; D000242:Cyclic AMP; D015056:1-Methyl-3-isobutylxanthine