Characterization of Ionotropic Glutamate Receptors in Rat Hypothalamus, Pituitary and Immortalized Gonadotropin-Releasing Hormone (GnRH) Neurons (GT1-7 Cells)

Abstract

Evidence from various sources suggested that the Gonadotropin-Releasing Hormone (GnRH) neuron does not contain glutamate receptors. Northern analysis of the hypothalamus showed the presence of NMDAR1, GluR1, GluR4 and GluR6 mRNA, while the pituitary showed the presence of NMDAR1, GluR1 and GluR6 mRNA. Western blot analysis also showed the presence of NMDAR1 and GluR1 protein. Since there are relatively few GnRH neurons in the hypothalamus, and GT1-7 cells have been considered to be a GnRH neuronal cell line, GT1-7 cells were studied in detail. GT1-7 cells contained NMDAR1 mRNA levels as shown by Northern analysis but did not contain GluR1, GluR4, or GluR6 mRNA. They did not show the presence of NMDAR1 and GluR1 protein by Western analysis. In addition, GT1-7 cells showed no NMDA receptor binding using the competitive inhibitor CGP-39563 and the noncompetitive inhibitor MK-801. Likewise, no binding was detected for kainate receptors. However, a small amount of binding for AMPA receptors was found in GT1-7 cells. GT1-7 cells did not exhibit glutamate toxicity and NMDA failed to elicit inward currents using patch-clamp techniques, although GABA did induce currents in the cells. As a whole, these studies suggest that GT1-7 cells lack or possess only low levels of ionotropic glutamate receptors.