Connexin 26 Expression and Extensive Gap Junctional Coupling in Cultures of GT1-7 Cells Secreting Gonadotropin-Releasing Hormone

Abstract

Gap junctions (GJs) are transmembrane channels that permit rapid intercellular transit of various small molecules including ions, second messengers and metabolites. GJs promote communication and coordinated activity between coupled neurons, and may help facilitate the synchronous release and pulsatile secretion of neurohormones. A previous study using GnRH-secreting GT1-7 cells reported that connexin 26 was the major GJ subunit present, and that about 20% of the cultured cells engaged in GJ coupling as assayed by fluorescence recovery after photobleaching of 5,6-carboxyfluorescein diacetate (MW 460 D). To reassess GJ connectivity with a more permeant probe, we grew GT1-7 cells to 70% confluency on Matrigel-coated glass coverslips and microinjected Neurobiotin^TM (MW 322 D) into single cells. Dye was allowed to diffuse for 30 min before cultures were fixed, and subsequently immunostained for Neurobiotin with 3,3′-diaminobenzidine HCl and examined by light microscopy. Dye coupling between 2 or more GT1-7 cells was observed after 75% of all microinjections. Connectivity involved the somata and neurites of an average of 6.6 ± 2.0 adjoining cells, but in one instance was seen in a group of 32 GT1-7 neighbors. Western blotting and immunofluorescence staining confirmed that connexin 26 was the predominant GJ subunit expressed by GT1-7 cultures. Our results using Neurobiotin suggest these GJ channels may be smaller than anticipated. In addition, functional GJ connectivity between subconfluent GT1-7 cells is more extensive than previously reported, occurring with higher frequency and coupling significantly greater numbers of cultured cells. Since cAMP, IP3, and Ca²⁺ are able to pass through GJs and can elicit secretion of GnRH by GT1 cell cultures, GJs may play an important role in the coordination and synchronization of GnRH release.